materials  rabbit antiserum against the bovine complex i 
  75-kda subunit was from prof .
the mouse monoclonal antibody against glutathionylated 
  proteins ( -gsh ) was from viro - gen corp .
[ s]gsh ( 942.0 ci / mmol ) was from 
  perkinelmer life sciences , and dithiothreitol ( dtt ) was removed prior to 
  experiments by extraction with ethyl acetate .
his - tagged grx2 was expressed 
  and isolated as described 
  ( 10 ) .
rabbit antiserum against human 
  sarcomeric mitochondrial creatine kinase was from abcam and cross - reacted with 
  the rat enzyme .
preparation of mitochondria , membranes , and complex i 
  bovine and rat heart mitochondria were prepared as described 
  ( 25 ) .
heart mitochondrial 
  preparations were stored on ice and used within 17 h of preparation 
  over which time the mitochondria remained well coupled .
bovine heart 
  mitochondrial membranes were prepared by disruption in a blender of 
  mitochondria , prepared as described 
  ( 26 ) , followed by isolation of 
  the membranes by centrifugation 
  ( 27 ) .
complex i was prepared 
  by solubilization of membranes with dodecyl -d - maltoside 
  ( ddm , anatrace , oh ) followed by ion - exchange chromatography 
  ( 28 ) .
pooled fractions were 
  further purified by gel filtration , and the purified complex i was stored in 
  buffer containing 0.1% ddm , 1 mm dtt , and 10% glycerol at -80 
  c . immediately prior to experiments , this buffer was replaced with one 
  lacking dtt by centrifugation in a micro bio - spin 6 chromatography column . 
 
glutathione assays  gsh , gssg , and protein - glutathione mixed 
  disulfides ( prssg ) were assayed using the recycling assay adapted for a 
  96-well plate reader 
  ( 2931 ) .
two - dimensional blue native ( bn)-page  for bn - page 
  ( 32 , 
  33 ) , mitochondria ( 0.5 mg of 
  protein ) were treated with 50 mm nem for 5 min , pelleted , and 
  resuspended in 60 l of extraction buffer : 1% ddm , 0.75 m 
  -amino - n - caproic acid ( aca ) , 50 mm bistris - hcl , ph 
  7.0 at 4 c .
after incubation on ice for 15 min , the suspension was 
  clarified by centrifugation in an airfuge ( beckman coulter ) at 17 
  p.s.i .
then 3.5 l of sample 
  buffer , comprising 5% ( w / v ) coomassie blue g 250 ( serva , germany ) and 500 
  mm aca , was added , and samples were resolved on a 1-mm - thick 
  512% acrylamide gradient gel containing 020% ( w / v ) glycerol , 0.5 
  m aca , 50 mm bistris - hcl , ph 7.0 ( 4 c ) , overlaid 
  with a 3.9% acrylamide stacking gel in the same buffer .
the anode buffer was 
  50 mm bistris , ph 7.0 , at 4 c , and the cathode buffer was 
  0.02% ( w / v ) coomassie blue g-250 , 50 mm tricine , 15 mm 
  bistris , ph 7.0 , at 4 c .
the gel was run at4 c for 1 h at 100 v and 
  then overnight at 40 v in cathode buffer without coomassie blue .
complex i 
  bands were then excised and incubated for 5 min at room temperature in 125 
  mm tris - hcl , ph 7.0 , 1% sds , and 50 mm nem before 
  insertion into the wells of a 3.7% acrylamide stacking gel in 0.13 
  m tris - hcl , ph 6.8 , overlaid on a 1-mm - thick sds - polyacrylamide 
  gel .
sds - page was done using either a 12.5% acrylamide linear gel , or a 
  520% acrylamide gradient gel containing 015% ( w / v ) sucrose and 
  0.375 m tris - hcl , ph 8.8 . stacking gel was then polymerized around 
  the excised bands and proteins separated by electrophoresis at 100120 v 
  in a miniprotean system ( bio - rad ) using 25 mm tris , 0.192 
  m glycine , 0.1% sds , ph 8.3 , as running buffer . immunoblotting
following sds - page , proteins were transferred 
  to a 0.2-m nitrocellulose membrane using a mini trans - blot system 
  ( bio - rad ) in transfer buffer comprising 48 mm tris , 39 
  mm glycine , 0.05% sds , 20% ( v / v ) methanol , ph 8.3 .
the membrane was 
  then blocked in pbst ( pbs , 0.05% ( v / v ) tween 20 with 1% ( w / v ) skimmed milk 
  powder ) and incubated with primary antiserum for 12 h at room 
  temperature ( gsh , 1:1000 ; ci75 , 1:1000 ; -creatine kinase 
  ( sarcomeric mitochondrial isoform ) , 1.25 g / ml ) .
blots were incubated with 
  1:50001:10,000 dilutions of the appropriate secondary antiserum for 1 h 
  at room temperature , treated with the chemiluminescent reagent ( ecl or ecl 
  plus , amersham biosciences ) according to the manufacturer 's instructions , and 
  visualized on super rx photographic film ( fujifilm ) . to visualize biotinylated 
  reagents
, blots were incubated with extravidin - peroxidase conjugate ( 1:1000 , 
  sigma ) instead of a primary antibody .
blots were re - probed by stripping in 
  62.5 mm tris - hcl ( ph 6.8 ) , 2% sds , 100 mm 
  -mercaptoethanol for 30 min at 50 c and then washed extensively in 
  pbst .
radiolabeled proteins were transferred from 
  gels to nitrocellulose as described above or the gel was dried onto filter 
  paper .
gels or blots were then exposed to a phosphor screen ( ge healthcare ) , 
  and the results were digitized with a typhoon detector ( ge healthcare ) .
protein spot excision and mass spectrometry  following 
  sds - page , protein bands were excised with a razor blade , transferred to a 
  0.5-ml tube that had been pre - washed with 50% methanol , and digested by 
   in - gel  cleavage 
  ( 34 ) . to do this
, the gel 
  slice was washed in high pressure liquid chromatography grade water ( 100 
  l ) for 30 min ; 20 mm tris - hcl , ph 7.0 ( 100 l ) for 30 min ; 
  100 l of 50% acetonitrile , 20 mm tris - hcl , ph 7.0 ( 100 l ) 
  for 30 min ; and 100% acetonitrile ( 20 l ) for 10 min .
the gel pieces were 
  then dried completely in a speedvac at 37 c for  20 min .
the gels 
  were rehydrated with 37 l of 20 mm tris - hcl , ph 7.0 , 5 
  mm cacl2 containing 12.5 ng/l trypsin ( roche applied 
  science ) or with 25 ng/l sequencing grade endoproteinase asp - n ( roche 
  applied science ) in 20 mm tris - hcl , ph 7.0 , and digested overnight 
  at 37 c .
all digests were examined in 
  a maldi - tof - tof mass spectrometer ( model 4700 proteomics analyzer , applied 
  biosystems ) using -cyano - hydroxy - trans - cinnamic acid as the 
  matrix . for trypsin digests ,
the instrument was calibrated with bovine trypsin 
  autolysis products ( m / z 2163.057 and 2273.160 ) and a calcium 
  related matrix ion ( m / z 1060.048 ) . for asp - n digestions , the 
  instrument was calibrated with pseudomonas fragi asp - n autolysis 
  product ( m / z 1723.743 ) and two complex i 75-kda asp - n lysis 
  products ( m / z 830.404 and 1329.753 ) .
peptide sequences were obtained by tandem ms in a maldi - tof - tof mass 
  spectrometer .
peptide masses and peptide fragmentation data were compared 
  against the national center for biotechnology information nr 20080103 data 
  base against all entries using mascot 
  ( 35 ) .
the parameters used were 
  as follows : peptide tolerance  25 ppm , ms / ms tolerance  0.8 
  da , allow for 1 missed cleavage and nem as a variable modification . ms and 
  tandem ms data were generally acquired automatically , and peptide 
  fragmentation spectra were interpreted automatically with the mascot ms - ms 
  web - based tool ( 35 ) .
when 
  glutathionylation on complex i was investigated , peaks from maldi - tof spectra 
  were identified manually in data explorer ( applied biosystems ) . 
 
identifications were subsequently supported by maldi - tof - tof data that were 
  analyzed manually and also using the ion fragmentation calculator feature in 
  data explorer software ( applied biosystems ) .
the digest containing the rat 
  cys-531 glutathionylated peptide was purified by capillary reverse phase 
  chromatography , and a portion was analyzed by maldi - tof - tof .
ros assays  to measure superoxide formation , bovine heart 
  mitochondrial membranes ( 1 mg protein / ml ) were first incubated with 1 
  mm dtt in kpi buffer ( 50 mm kpi , 
  ph 8 , 1 mm egta , 100 m dtpa , 100 m 
  neocuproine ) at 37 c for 10 min , pelleted by centrifugation ( 14,000 
   g for 7 min ) , and then washed twice in 0.5 ml of 
  kpi buffer .
membranes ( 0.2 mg of protein / ml ) were then incubated in 
  kpi buffer at 30 c with 2 m coelenterazine 
  ( clz ) in a luminometer ( berthold auto - lumatplus lb 953 ) with further 
  additions as indicated in the figure legends , and the chemiluminescence was 
  recorded for 5 s every 30 s over a 5-min incubation 
  ( 25 , 
  36 ) .
chemiluminescence was 
  stable for the duration of the incubation , and the average chemiluminescence 
  was recorded .
superoxide production by heart mitochondria was measured by two methods as 
  follows : clz chemiluminescence 
  ( 25 , 
  36 ) and by dihydroethidine 
  oxidation ( 37 , 
  38 ) . for the chemiluminescence 
  measurements ,
mitochondria ( 100 g of protein / ml ) were incubated 1 
  mm diamide in 120 mm kcl , 10 mm hepes , 1 
  mm edta , 100 m dtpa , ph 7.2 , supplemented with 2 
  m clz , and 5 mm succinate with 8 g / ml rotenone 
  or 10 mm glutamate / malate were used as substrates .
clz 
  chemiluminescence was measured as described above . for the dihydroethidine 
  assay , mitochondria ( 0.5 mg of protein / ml )
were incubated in 120 mm 
  kcl , 10 mm hepes , 1 mm edta , 100 m dtpa , 
  ph 7.2 , and 10 m dihydroethidine .
succinate ( 10 mm ) 
  and 4 g / ml rotenone or 10 mm glutamate / malate were used as 
  respiratory substrates .
the mitochondria were incubated 1 
  mm diamide or 2.5 m antimycin for 5 min , and 
  then 0.1% triton x-100 was added followed 1 min later by 50 g / ml salmon 
  sperm dna .
the fluorescence of the dihydroethidine oxidation products 
  intercalated into dna was measured in a 3-ml stirred cuvette at 30 c in a 
  shimadzu rf-5301 fluorimeter ( ex 520 nm , 
  em 590 nm ) .
control experiments showed that this amount of 
  dna was sufficient to maximize the fluorescence of 10 m 
  ethidium bromide and that the presence of diamide did not quench the 
  fluorescence .
this assay assesses the production of both 2-hydroxyethidium and 
  ethidium , whereas production of 2-hydroethidium is specific for superoxide , 
  there are other pathways to the formation of ethidium under conditions of 
  oxidative stress ( 37 , 
  38 ) .
general assays  to measure the effect of diamide on enzyme 
  activities , rat heart mitochondria ( 0.5 mg of protein / ml ) were incubated in 
  kcl buffer ( 120 mm kcl , 10 mm hepes , 1 mm 
  egta , ph 7.2 or 7.5 ) at 37 c for 5 min in the presence of 10 
  mm glutamate / malate and then pelleted by centrifugation .
complex i 
  activity was measured using 2040 g of protein / ml at 30 c as 
  the rotenone - sensitive oxidation of nadh ( 340 = 6.22 
  mmcm ) in 50 mm kcl , 1 
  mm edta , 10 mm tris - hcl , ph 7.4 , supplemented with 300 
  nm antimycin , 100 m nadh , and 50 m 
  decylubiquinone containing phosphatidylcholine ( 10 mg / ml ) 
  ( 39 ) .
complex ii / iii activity 
  was measured in 40 mm kpi , 0.5 mm edta , ph 
  7.4 , 20 mm succinate , and 2 mm kcn using 20 g of 
  protein / ml at 30 c .
after 10 min pre - equilibration 30 m 
  cytochrome c was added and its rate of reduction measured 
  ( 550 = 21 mmcm ) . 
 
respiration rates were measured using a clark - type oxygen 
  electrode ( rank brothers , bottisham , cambridge , uk ) .
rat heart mitochondria 
  ( 12 mg protein / ml ) were incubated in the stirred 3-ml electrode chamber 
  at 30 c in 120 mm kcl , 10 mm hepes , 1 mm 
  egta , ph 7.2 , in the presence of 0 , 0.5 , or 1 mm diamide . after 2 
  min
, 10 mm succinate in the presence of 4 g / ml rotenone or 10 
  mm glutamate / malate was added , and the rate of coupled respiration 
  was measured . after a further 23 min , carbonyl cyanide 
  4-(trifluoromethoxy)phenylhydrazone ( fccp ; 1
protein concentration was 
  determined by the bicinchoninic acid assay using bovine serum albumin as a 
  standard ( 40 ) .
multiple sequence alignments and comparative modeling of the complex i 
  75-kda subunit  orthologous protein sequences of the subunits of 
  complex i from thermus thermophilus were identified in the uniprot 
  resource using blastp ( 41 ) . 
 
sequences were aligned using clustalw 
  ( 42 ) with the t. 
  thermophilus structure used to weight the gap penalties for the 
  preferential placement of gaps in loop regions , rather than secondary 
  structure elements .
a set of nonredundant sequences was selected from diverse 
  organisms to determine the extent of residue conservation across members of 
  the protein family , including t. thermophilus , deinococcus radiodurans , 
  reclinomonas americana , bos taurus , drosophila melanogaster , caenorhabditis 
  elegans , neurospora crassa , arabidopsis thaliana , phytophthora infestans , 
  acanthamoeba castellani , and dictyostelium discoideum .
the 
  extent of residue conservation in species more closely related to b. 
  taurus was determined from a set , including protein sequences from 
  mammals , gallus gallus , xenopus laevis , and danio rerio .
the 
  alignment of the protein sequences for the bovine and t. thermophilus 
  complex i 75-kda subunits was used to identify the location in the bovine 
  protein of the non - iron - sulfur center cys residues in the structure .
the 
  comparative protein modeling program modeller 
  ( 43 ) was used to calculate a 
  model of subunits of bovine complex i that are orthologous to those of t. 
  thermophilus , by using the pairwise sequence alignments .
pymol was used 
  to visualize and analyze the protein structures and the local environment of 
  residues .
complex i 75-kda subunit is glutathionylated in oxidatively stressed 
  mitochondria  to induce oxidative stress , we exposed heart 
  mitochondria to the thiol oxidant diamide , which converted gsh to gssg and led 
  to the formation of protein - mixed disulfides with glutathione ( prssg ) by 
  thiol - disulfide exchange ( fig . 
 
to visualize glutathionylated mitochondrial 
  proteins , we lysed mitochondria in the presence of the thiol alkylating 
  reagent nem to prevent transglutathionylation , separated the proteins by 
  nonreducing sds - page , and probed with an antibody against glutathionylated 
  proteins ( fig .
diamide treatment led to the glutathionylation of a 
  number of proteins that were reversed by the thiol reductant dtt 
  ( fig .
reprobing with antiserum against the complex i 75-kda 
  subunit showed that this subunit co - migrated with a glutathionylated protein 
  ( fig .
figure 1.glutathionylation of the 75-kda subunit complex i in oxidatively 
  stressed mitochondria . a , rat heart mitochondria ( 1 mg of 
  protein / ml ) were incubated in kcl buffer with 10 mm 
  glutamate / malate for 5 min at 37 c  0.5 mm diamide . 
 
control mitochondria contained 1.0  0.3 nmol 
  of gsh equivalents (= gsh + 2gssg + prssg)/mg of protein ( n = 3 
   s.e . ) .
recovery of gsh equivalents after diamide treatment was 
  64% because of gsh loss from oxidatively stressed mitochondria and/or 
  oxidation of gsh to higher oxidation states than gssg .
b , 
  mitochondria were treated as in a and then resuspended in loading 
  buffer containing 50 mm nem ( -dtt ) or 100 mm 
  dtt ( + dtt ) , and 50 g of protein was separated by sds - page and 
  electrotransferred to nitrocellulose .
glutathionylated proteins were 
  visualized using a specific antibody ( gsh ) , and the blot was 
  then re - probed with an antibody against the complex i 75-kda subunit antibody 
  ( ci75 ) .
samples were resolved on a single gel separated by 
  empty lanes to prevent dtt leaching .
these empty lanes have been removed , and 
  a gray vertical line indicates where the two parts of the blots have 
  been rejoined .
the arrow indicates a minor glutathionylated protein 
  that co - migrates with the 75-kda subunit .
c , mitochondria ( 1 mg of 
  protein / ml ) were incubated  diamide as in a , and then treated 
  10 mm dtt for 10 min at 37 c followed by addition of 
  50 mm nem and separation by bn - page .
the complex i band was then 
  separated by nonreducing sds - page , electrotransferred to nitrocellulose , and 
  probed with an antibody against glutathionylated proteins .
d , 
  mitochondria were incubated  diamide as in a and then 
  glutathionylation of complex i was assessed by bn - page followed by nonreducing 
  sds - page as in c ( gsh ) .
the blot was then re - probed 
  with an antibody against the 75-kda complex i subunit ( ci75 ) . 
 
e , mitochondria ( 20 mg of protein / ml ) were preincubated with the 
  biotinylated ethyl ester of glutathione ( 1.42 mm ) for 30 min on 
  ice , pelleted , and washed in 250 mm sucrose , 1 mm egta , 
  10 mm tris - hcl , ph 7.4 .
the mitochondria were then incubated 
   0.5 mm diamide as in a , and biotinylation of 
  complex i was assessed following bn - page and nonreducing sds - page as in 
  c by probing with extravidin .
the blot was then re - probed with an 
  antibody against the complex i 75-kda subunit antibody ( ci75 ) . 
 
f , mitochondria ( 20 mg of protein / ml ) were first incubated with 
  [ s]gsh ( 40 ci / ml ; 37 nm ) in ste for 30 min on ice , 
  pelleted , and washed .
the [ s]gsh - loaded mitochondria were then 
  incubated  diamide , separated , and electrotransferred onto 
  nitrocellulose as in c , and [ s]gsh - containing protein 
  bands were detected using a phosphorimager .
the blot was then re - probed with 
  an antibody against the complex i 75-kda subunit ( ci75 ) . 
 
g , [ s]gsh - loaded mitochondria were incubated  
  diamide , and complex i was separated by bn - page followed by nonreducing 
  sds - page as in f. the gel was dried , and 
  [ s]gsh - containing protein bands were detected using a 
  phosphorimager ( [ s]gsh ) .
two bands labeled by [ s]gsh 
  ( gel , bands a and b ) were excised , and identification was 
  attempted by peptide mass fingerprinting .
h , band a from g 
  above was analyzed by peptide mass fingerprinting following in - gel digestion 
  with trypsin .
additional evidence for the identification of the 75-kda subunit is 
  provided by the analysis of three peptides by tandem ms ( maldi - tof - tof ) .
there was no 
  significant identification for band b. glutathionylation of the 75-kda subunit complex i in oxidatively 
  stressed mitochondria .
a , rat heart mitochondria ( 1 mg of 
  protein / ml ) were incubated in kcl buffer with 10 mm 
  glutamate / malate for 5 min at 37 c  0.5 mm diamide . 
 
control mitochondria contained 1.0  0.3 nmol 
  of gsh equivalents (= gsh + 2gssg + prssg)/mg of protein ( n = 3 
   s.e . ) .
recovery of gsh equivalents after diamide treatment was 
  64% because of gsh loss from oxidatively stressed mitochondria and/or 
  oxidation of gsh to higher oxidation states than gssg .
b , 
  mitochondria were treated as in a and then resuspended in loading 
  buffer containing 50 mm nem ( -dtt ) or 100 mm 
  dtt ( + dtt ) , and 50 g of protein was separated by sds - page and 
  electrotransferred to nitrocellulose .
glutathionylated proteins were 
  visualized using a specific antibody ( gsh ) , and the blot was 
  then re - probed with an antibody against the complex i 75-kda subunit antibody 
  ( ci75 ) .
samples were resolved on a single gel separated by 
  empty lanes to prevent dtt leaching .
these empty lanes have been removed , and 
  a gray vertical line indicates where the two parts of the blots have 
  been rejoined .
the arrow indicates a minor glutathionylated protein 
  that co - migrates with the 75-kda subunit .
c , mitochondria ( 1 mg of 
  protein / ml ) were incubated  diamide as in a , and then treated 
  10 mm dtt for 10 min at 37 c followed by addition of 
  50 mm nem and separation by bn - page .
the complex i band was then 
  separated by nonreducing sds - page , electrotransferred to nitrocellulose , and 
  probed with an antibody against glutathionylated proteins .
d , 
  mitochondria were incubated  diamide as in a and then 
  glutathionylation of complex i was assessed by bn - page followed by nonreducing 
  sds - page as in c ( gsh ) .
the blot was then re - probed 
  with an antibody against the 75-kda complex i subunit ( ci75 ) . 
 
e , mitochondria ( 20 mg of protein / ml ) were preincubated with the 
  biotinylated ethyl ester of glutathione ( 1.42 mm ) for 30 min on 
  ice , pelleted , and washed in 250 mm sucrose , 1 mm egta , 
  10 mm tris - hcl , ph 7.4 .
the mitochondria were then incubated 
   0.5 mm diamide as in a , and biotinylation of 
  complex i was assessed following bn - page and nonreducing sds - page as in 
  c by probing with extravidin .
the blot was then re - probed with an 
  antibody against the complex i 75-kda subunit antibody ( ci75 ) . 
 
f , mitochondria ( 20 mg of protein / ml ) were first incubated with 
  [ s]gsh ( 40 ci / ml ; 37 nm ) in ste for 30 min on ice , 
  pelleted , and washed .
the [ s]gsh - loaded mitochondria were then 
  incubated  diamide , separated , and electrotransferred onto 
  nitrocellulose as in c , and [ s]gsh - containing protein 
  bands were detected using a phosphorimager .
the blot was then re - probed with 
  an antibody against the complex i 75-kda subunit ( ci75 ) . 
 
g , [ s]gsh - loaded mitochondria were incubated  
  diamide , and complex i was separated by bn - page followed by nonreducing 
  sds - page as in f. the gel was dried , and 
  [ s]gsh - containing protein bands were detected using a 
  phosphorimager ( [ s]gsh ) .
two bands labeled by [ s]gsh 
  ( gel , bands a and b ) were excised , and identification was 
  attempted by peptide mass fingerprinting .
h , band a from g 
  above was analyzed by peptide mass fingerprinting following in - gel digestion 
  with trypsin .
additional evidence for the identification of the 75-kda subunit is 
  provided by the analysis of three peptides by tandem ms ( maldi - tof - tof ) .
there was no 
  significant identification for band b. to analyze complex i , the intact protein was isolated from oxidatively 
  stressed mitochondria by bn - page and then dissociated into its subunits by 
  nonreducing sds - page ( fig . 
 
an antibody against glutathionylated proteins 
  revealed a single , dtt - sensitive band present in diamide - treated but not 
  control samples ( fig . 
 
reanalysis with antiserum against the bovine complex 
  i 75-kda subunit showed that the glutathionylated protein co - migrated with the 
  75-kda subunit ( fig . 
 
although sds - page used in the second dimension may 
  not resolve all complex i polypeptides 
  ( 44 , 
  45 ) , the full complement of 
  complex i subunits should be present .
protein glutathionylation was analyzed 
  by an alternative method using the membrane - permeant biotinylated ethyl ester 
  of glutathione .
after incubation with this glutathione derivative , the 
  mitochondria were washed and treated with diamide , and then complex i was 
  isolated as before by bn - page followed by nonreducing sds - page 
  ( fig .
diamide 
  treatment led to the dtt - sensitive binding of biotinylated glutathione to a 
  protein that co - migrated with the complex i 75-kda subunit 
  ( fig .
to further 
  investigate glutathionylation , we preincubated mitochondria with 
  [ s]gsh ( 46 ) and 
  after washing exposed them to diamide .
complex i was isolated by bn - page , and 
  following nonreducing sds - page , [ s]gsh binding was assessed 
  ( fig .
this showed 
  a dtt - sensitive [ s]gsh band at about 75-kda that co - migrated with 
  the complex i 75-kda subunit ( fig . 
 
another [ s]gsh - labeled band at 45 
  kda was not detected using the gsh antibody or biotinylated glutathione 
  ester . to identify the [ s]gsh - labeled proteins ,
the gel was 
  dried , and [ s]gsh - containing bands were visualized , and the bands 
  at 75 and 45 kda were excised 
  ( fig .
peptide 
  mass fingerprinting and tandem mass spectrometry confirmed that one band was 
  the complex i 75-kda subunit ( fig . 
 
no identification was made of the 
  [ s]gsh - containing band at 45 kda ; however , other experiments 
  ( supplemental fig .
1 ) suggested that it was sarcomeric creatine kinase , a 
  protein that is known to become glutathionylated 
  ( 47 ) .
figure 2.amino acid sequences of the mature bovine complex i 75-kda subunit . 
  the primary sequence of mature bovine complex i 75-kda subunit is shown 
  ( p15690 ) .
the location of the cys residues is the same in the corresponding 
  rat sequence ( q66hf1 ) .
the cysteine - containing peptides identified by 
  maldi - tof following proteolysis of complex i are marked by gray 
  boxes .
the 11 cys residues involved in iron - sulfur centers are shown in 
  yellow typeface .
the six cys residues that are not involved in 
  iron - sulfur centers are in red and are surrounded by yellow 
  boxes .
amino acid sequences of the mature bovine complex i 75-kda subunit . 
  the primary sequence of mature bovine complex i 75-kda subunit is shown 
  ( p15690 ) .
the location of the cys residues is the same in the corresponding 
  rat sequence ( q66hf1 ) .
the cysteine - containing peptides identified by 
  maldi - tof following proteolysis of complex i are marked by gray 
  boxes .
the 11 cys residues involved in iron - sulfur centers are shown in 
  yellow typeface .
the six cys residues that are not involved in 
  iron - sulfur centers are in red and are surrounded by yellow 
  boxes .
previous work showed glutathionylation of the 75- and 51-kda subunits of 
  purified complex i on incubation with gssg 
  ( 911 , 
  17 ) .
however , when gssg 
  binding to purified complex i was investigated using low concentrations of 
  radiolabeled or biotinylated gsh , the 75-kda subunit predominated , along with 
  a number of other minor bands , whereas higher gssg concentrations were 
  required for glutathionylation of the 51-kda subunit ( data not shown ) .
as 
  incubation of purified complex i with gssg leads to artifactual 
  glutathionylation because of denaturation of the complex 
  ( 9 ) , glutathionylation of the 
  51-kda subunit may only occur in vitro .
our findings confirm that 
  complex i is glutathionylated on the 75-kda subunit within oxidatively 
  stressed mitochondria and suggest that this is the only complex i subunit 
  glutathionylated .
identification of the cysteine residues glutathionylated on the 75-kda 
  subunit of purified complex i  the bovine ( p15690 ) and rat ( q66hf1 ) 
  complex i 75-kda subunits contain a 23-amino acid n - terminal targeting 
  sequence that is removed after mitochondrial import leaving 704 amino acid 
  polypeptides that are 94.8% identical 
  ( 48 ) .
the mature 75-kda 
  polypeptides contain 17 cys residues , with 11 ( cys-41 , -52 , -54 , -69 , -105 , 
  -108 , -114 , -153 , -156 , -159 , and -203 ) involved in the 75-kda subunit three 
  iron - sulfur centers ( fig .
2 ) 
  ( 2 , 
  4 ) . the remaining six cys 
  residues ( cys-30 , -344 , -531 , -541 , -687 , and -704 ) are potential candidates 
  for glutathionylation and are shown in red in 
  fig . 2 . to determine whether mass spectrometry could be used to reliably assess 
  glutathionylation of these cys residues , purified bovine complex i was reduced 
  and denatured in sds , and protein thiols were alkylated with nem , and the 
  75-kda subunit was separated by nonreducing sds - page . following in - gel 
  proteolysis with asp - n or trypsin , peptide masses were measured by 
  maldi - tof - ms ( table 1 ) , and 
  their sequences were confirmed by tandem mass spectrometry in a maldi - tof - tof 
  ( supplemental table 1 ) .
we detected 12 of the 15 cys - containing peptides in 
  their nem - modified forms , accounting for 13 of the 17 cys residues 
  ( table 1 ) .
the four cys 
  residues not detected were all involved in the coordination of iron - sulfur 
  centers ( table 1 ) , and 
  consequently were unlikely candidates for glutathionylation .
table 1maldi - tof analysis of nem - alkylated , cys - containing peptides from the 
  bovine and rat complex i 75-kda subunit experimental masses were obtained 
  from isolated and denatured complex i ( 2 mg / ml ) treated with dtt ( 10 
  mm ) and sds ( 1% ) for 10 min at 37c ; the dtt was removed ; and 
  the samples were treated with nem .
bovine or rat mitochondria ( 1 mg of 
  protein / ml ) were incubated with diamide ( 0.5 mm ) for 5 min at 
  37c .
after incubation all samples were alkylated with nem ( 2550 
  mm ) and separated by electrophoresis ( one - dimensional sds - page for 
  isolated complex i samples and two - dimensional bn / sds - page for mitochondria ) 
  before in - gel digestion and maldi-tof.cys 
  no.sequenceenzymefesmass of nem - alkylated peptide ( mh)calculatedobserveddenatured complex ibovine mitochondria ( b)rat mitochondria ( r ) 30 
   tataasnlievfvdgqsvmvepgttvlqacek ( b ) 
   t 
   no 
   3433.6865 
   3433.6977 
   3433.6301 
   tgtaasnlievfvdgqsvmvepgttvlqacek ( r ) 
   3419.6707 
   3419.7310 
   41 
   fcyher 
   t 
   yes 
   979.4090 
   979.4006 
   979.4030 
   979.4123 
   52 
   lsvagncr 
   t 
   yes 
   944.4618 
   944.4646 
   944.4555 
   944.4660 
   55 
   mclveiek 
   t 
   yes 
   1089.5319 
   1089.5171 
   1089.5236 
   1089.5527 
   69 
   vvaacampvmk 
   t 
   yes 
   1244.6200 
   1244.6146 
   1244.6105 
   1244.6212 
   105,108 
   dcpic or dcpic d 
   yes 
   675.2475 
   dcpic 800.2945 
   114 
  
dqggec d 
   yes 
   733.2456 
   153,156 
  ciqctr or ciqctr 
   t 
   yes 
   848.3753 
   848.3709 
   848.3825 
   848.3458 
  ciqctr 
   973.4230 
   973.4194 
   973.4202 
   973.4334 
   159 
 
cir 
   t 
   yes 
   516.2597 
   203 
   dicpvgaltskpyaftarpw 
   d 
   yes 
   2318.1564 
   2318.1790 
   2318.2058 
   2318.2073 
   344 
   vdsdtlcteevfptagagtdlr ( b ) 
   t 
   no 
   2422.1078 
   2422.0918 
   2422.0857 
   vdsdtlcteeifpnegagtdlr ( r ) 
   2507.1241 
   2507.1228 
   531 
   mlfllgadggcitr ( b ) 
   t 
   no 
   1591.7970 
   1591.7957 
   1591.7982 
   llfllgadggcitr ( r ) 
   1573.8405 
   1573.8358 
   541 
   dcfivyqghhg 
   d 
   no 
   1400.5979 
   1400.5907 
   1400.6039 
   1400.6027 
   687 
   dsisrasqtmakcvkavtegahave ( b ) 
   d 
   no 
   2714.3163 
   2714.3508 
   2714.3940 
   dsisrasqtmakcvkavtegaqave ( r ) 
   2705.3231 
   2705.3904 
   704 
   avtegahaveepsic ( b ) 
   t 
   no 
   1637.7475 
   1637.7476 
   1637.7484 
 
  avtegaqaveepsic ( r ) ( nem ) 
 
  1503.6995 
 
  1503.6367 
  acysteine residue numbers are shown , which are the same for bovine and rat 
  sequencesbunless specified as bovine ( b ) or rat ( r ) , peptide sequences are the same 
  for both speciesct indicates trypsin , and d indicates asp - nddata indicate whether the cysteine residue is part of an iron - sulfur 
  clusterecalculated masses of peptides were alkylated with nem ( + 125.047 da ) maldi - tof analysis of nem - alkylated , cys - containing peptides from the 
  bovine and rat complex i 75-kda subunit experimental masses were obtained 
  from isolated and denatured complex i ( 2 mg / ml ) treated with dtt ( 10 
  mm ) and sds ( 1% ) for 10 min at 37c ; the dtt was removed ; and 
  the samples were treated with nem .
bovine or rat mitochondria ( 1 mg of 
  protein / ml ) were incubated with diamide ( 0.5 mm ) for 5 min at 
  37c .
after incubation all samples were alkylated with nem ( 2550 
  mm ) and separated by electrophoresis ( one - dimensional sds - page for 
  isolated complex i samples and two - dimensional bn / sds - page for mitochondria ) 
  before in - gel digestion and maldi - tof .
cysteine residue numbers are shown , which are the same for bovine and rat 
  sequences unless specified as bovine ( b ) or rat ( r ) , peptide sequences are the same 
  for both species t indicates trypsin , and d indicates asp - n data indicate whether the cysteine residue is part of an iron - sulfur 
  cluster calculated masses of peptides were alkylated with nem ( + 125.047 da ) to see if glutathionylated peptides could be detected , we reduced complex i 
  with dtt and then denatured it with sds . after removing the dtt , the denatured 
  complex was incubated with 20 mm gssg for 30 min , followed by nem 
  to prevent subsequent transglutathionylation .
after nonreducing sds - page and 
  proteolytic digestion , the cys - containing peptides from the 75-kda subunit 
  were analyzed by maldi - tof - ms for a + 305 m / z shift 
  consistent with glutathionylation ( table 
  2 ) .
eight of the 15 peptides containing cys residues were detected 
  as glutathionylated peptides ( table 
  2 ) , and in all cases glutathionylation was reversed by treatment 
  with dtt ( data not shown ) .
these cys residues included all six of the 
  non - iron - sulfur center cys residues .
glutathionylation of cys-41 and cys-203 , 
  which are components of iron - sulfur centers , was also detected , suggesting 
  that these residues became available for modification on denaturation of 
  purified complex i. table 2maldi - tof analysis of glutathionylated , cys - containing peptides from the 
  bovine and rat complex i 75 kda subunit experimental masses were obtained 
  from isolated and denatured complex i ( 2 mg / ml ) and treated with dtt ( 10 
  mm ) and sds ( 1% ) for 10 min at 37c ; the dtt was removed ; and 
  the samples were treated with gssg ( 20 mm ) for 30 min at 37c . 
  for native complex
i , isolated bovine complex i was incubated with gssg ( 20 
  mm ) for 30 min at 37c ; bovine or rat mitochondria ( 1 mg of 
  protein / ml ) were incubated with diamide ( 0.5 mm ) for 5 min at 
  37c .
after incubation all samples were alkylated with nem ( 2550 
  mm ) and separated by electrophoresis ( one - dimensional sds - page for 
  isolated ci samples and two - dimensional bn / sds - page for mitochondria ) before 
  in - gel digestion and maldi-tof.cys 
  no.sequenceenzymefesmass ( mh ) + gssg ( 305.068)calculatedobserveddenatured complex inative complex ibovine mitochondria ( b)rat mitochondria ( r ) 30 
   tataasnlievfvdgqsvmvepgttvlqacek ( b ) 
   t 
   no 
   3613.7074 
   3613.7356 
   tgtaasnlievfvdgqsvmvepgttvlqacek ( r ) 
   3559.6917 
   41 
   fcyher 
   t 
   yes 
   1159.4295 
   1159.4165 
   52 
   lsvagncr 
   t 
   yes 
   1124.4822 
   55 
   mclveiek 
   t 
   yes 
  
1269.5529 
   69 
   vvaacampvmk 
   t 
   yes 
   1424.6405 
   105,108 
   dcpic or dcpic d 
   yes 
   980.3164 
   dcpic 1160.3368 
   114 
   dqggec d 
   yes 
   913.2668 
   153,156 
  ciqctr or ciqctr 
   t 
   yes 
   1403.5430 
  ciqctr 
   1583.5630 
   159 
  cir 
   t 
   yes 
   696.2809 
   203 
   dicpvgaltskpyaftarpw 
   d 
   yes 
   2498.1842 
   2498.1648 
   2498.1743 
   344 
   vdsdtlcteevfptagagtdlr ( b ) 
   t 
   no 
   2602.1135 
   2602.1125 
   vdsdtlcteeifpnegagtdlr ( r ) 
   2687.1451 
   531 
   mlfllgadggcitr ( b ) 
   t 
   no 
   1771.8175 
   1771.8016 
   1771.8175 
   1771.8191 
   llfllgadggcitr ( r ) 
   1753.8453 
   1753.8760 
   541 
   dcfivyqghhg 
  
d 
   no 
   1580.6261 
   1580.6172 
   1580.6201 
   687 
   dsisrasqtmakcvkavtegahave ( b ) 
   d 
   no 
   2894.3440 
   2894.3035 
   2894.3816 
   dsisrasqtmakcvkavtegaqave ( r ) 
   2885.3441 
   704 
   avtegahaveepsic ( b ) 
   t 
   no 
   1817.7680 
   1817.7848 
   1817.7869 
   1817.7720 
 
  avtegaqaveepsic ( r ) 
 
  1808.7681 
  acysteine residue numbers are shown , which are the same for bovine and rat 
  sequencesbunless specified as bovine ( b ) or rat ( r ) , peptide sequences are the same 
  for both speciesct indicates trypsin , and d indicates asp - nddata indicate whether the cysteine residue is part of an iron - sulfur 
  clusterecalculated masses of peptides were glutathionylated on the cys residues 
  ( + 305.068 da ) maldi - tof analysis of glutathionylated , cys - containing peptides from the 
  bovine and rat complex i 75 kda subunit experimental masses were obtained 
  from isolated and denatured complex i ( 2 mg / ml ) and treated with dtt ( 10 
  mm ) and sds ( 1% ) for 10 min at 37c ; the dtt was removed ; and 
  the samples were treated with gssg ( 20 mm ) for 30 min at 37c . 
  for native complex
i , isolated bovine complex i was incubated with gssg ( 20 
  mm ) for 30 min at 37c ; bovine or rat mitochondria ( 1 mg of 
  protein / ml ) were incubated with diamide ( 0.5 mm ) for 5 min at 
  37c .
after incubation all samples were alkylated with nem ( 2550 
  mm ) and separated by electrophoresis ( one - dimensional sds - page for 
  isolated ci samples and two - dimensional bn / sds - page for mitochondria ) before 
  in - gel digestion and maldi - tof .
cysteine residue numbers are shown , which are the same for bovine and rat 
  sequences unless specified as bovine ( b ) or rat ( r ) , peptide sequences are the same 
  for both species t indicates trypsin , and d indicates asp - n data indicate whether the cysteine residue is part of an iron - sulfur 
  cluster calculated masses of peptides were glutathionylated on the cys residues 
  ( + 305.068 da ) next , glutathionylation of cys residues in native complex i was 
  investigated by incubating intact complex i with 20 mm gssg 
  followed by nem , and then protease digests of the 75-kda subunit were analyzed 
  by mass spectrometry .
five cys residues ( cys-203 , -531 , -541 , -687 , and -704 ) 
  were glutathionylated in the native complex .
cys-203 is involved with 
  iron - sulfur center ligation , suggesting some denaturation of the complex on 
  incubation with gssg .
when intact complex i was exposed to a lower 
  concentration of gssg ( 5 mm ) only three cys residues 
  ( cys531 , -687 , and -704 ) were glutathionylated ( data not shown ) .
this 
  identifies cys residues 531 , 687 , and 704 of the 75-kda subunit as plausible 
  candidates for glutathionylation within intact mitochondria .
recently cys-344 ( cys-367 in the precursor sequence ) of purified bovine 
  complex i 75-kda subunit was reported to be glutathionylated on exposure to 
  gssg ( 17 ) .
we found that this 
  residue was only glutathionylated in denatured , purified complex i and that it 
  was not glutathionylated in native , purified complex i or in diamide - treated 
  mitochondria , nor was there decreased recovery of nem - alkylated cys-344 . in 
  ref .
17 , the sequence coverage 
  of the 75-kda subunit was limited so that cys-344 was the only non - iron - sulfur 
  center cys residues investigated , so glutathionylation of the other five cys 
  residues would not have been detected 
  ( 17 ) .
it is possible that the 
  purified complex i preparations used were somewhat denatured , giving a 
  glutathionylation pattern similar to that of our sds - treated complex i 
  preparations ( table 2 ) .
identification of glutathionylated peptides from the 75-kda subunit of 
  complex i within oxidatively stressed mitochondria to assess 
  glutathionylation within mitochondria of the 75-kda subunit , bovine and rat 
  heart mitochondria were incubated  diamide , then the remaining free 
  cys residues were blocked with nem , and complex i was isolated by bn - page 
  followed by nonreducing sds - page .
peptides from the 75-kda subunit were 
  analyzed for nem and glutathione modification by maldi - tof - ms ( tables 
  1 and 
  2 ) , with confirmation of the 
  peptide sequences by tandem mass spectrometry ( supplemental table 1 ) . in the absence of diamide , 12 of the 15 cys - containing peptides in the 
  75-kda subunit of complex
after treatment of bovine mitochondria with diamide , analysis of digests of 
  the 75-kda subunit revealed that peptides containing cys-531 and cys-704 were 
  glutathionylated ( table 2 ) . 
 
however , glutathionylation of cys-704 in that rat 75-kda 
  subunit was not detected , although the amount of peptide containing unmodified 
  cys-704 was decreased by diamide .
figure 3.analysis by mass spectrometry of glutathionylated peptides from the 
  complex i 75-kda subunit in oxidatively stressed mitochondria .
bovine 
  heart mitochondria were incubated  diamide ; the 75-kda subunit of 
  complex i was then isolated by bn - page followed by nonreducing sds - page as 
  described in fig .
, the peptides containing cys-531 and cys-704 were analyzed 
  for glutathionylation by maldi - tof - ms as described in 
  table 2 .
an 8-da portion of the 
  mass spectra from the analysis of the modified peptides is shown .
data shown 
  for + diamide and - diamide traces are typical maldi - tof 
  traces , repeated at least three times with the same result .
analysis by mass spectrometry of glutathionylated peptides from the 
  complex i 75-kda subunit in oxidatively stressed mitochondria .
bovine 
  heart mitochondria were incubated  diamide ; the 75-kda subunit of 
  complex i was then isolated by bn - page followed by nonreducing sds - page as 
  described in fig .
, the peptides containing cys-531 and cys-704 were analyzed 
  for glutathionylation by maldi - tof - ms as described in 
  table 2 .
an 8-da portion of the 
  mass spectra from the analysis of the modified peptides is shown .
data shown 
  for + diamide and - diamide traces are typical maldi - tof 
  traces , repeated at least three times with the same result .
portions of typical maldi - tof mass spectra for peptides containing cys-531 
  or cys-704 from bovine heart mitochondria incubated  diamide are shown 
  in fig .
3 . in the presence of 
  diamide there are significant peaks at m / z 1771.8 and at 
  m / z 1817.8 ( fig . 
 
3 ) , corresponding to monoglutathionylation of the peptides 
  containing cys-531 and cys-704 , respectively . in mitochondria that had not 
  been incubated with diamide , there was no detectable glutathionylation of 
  peptides containing these two cys residues , and subsequent dtt treatment 
  removed these glutathionylated peaks ( fig . 
 
these data are consistent with glutathionylation of cys-531 
  and cys-704 within oxidatively stressed bovine heart mitochondria .
location of modified cysteine residues within glutathionylated 
  peptides  glutathionylated peptides containing cys-531 and cys-704 
  from diamide - treated mitochondria were analyzed by tandem mass spectrometry 
  ( maldi - tof - tof ) to determine the sites of glutathione modification . in 
  addition to the conventional b and y ion series ,
further fragmentations 
  adjacent to the disulfide bond of the glutathionylated cys residues were 
  observed and are illustrated in fig . 
 
fragmentation of the 1771.8-da peptide , corresponding to glutathionylation of 
  the bovine cys-531 peptide , gave a y - type ion series for y4 and 
  above , consistent with glutathionylation of cys-531 
  ( fig .
additional 
  y - type ions were present consistent with fragmentation at an amide bond within 
  the glutathione adduct or on either side of the disulfide bond linking the 
  adduct to cys-531 ( fig . 
 
fragmentation of the 1753.8 peptide , 
  corresponding to glutathionylation of the rat cys-531 peptide , also showed a 
  y series consistent with glutathionylation of cys-531 ( data not 
  shown ) .
fragmentation of the 1817.8-da peptide , corresponding to 
  glutathionylation of the bovine cys-704 peptide , gave a y series 
  consistent with glutathionylation of cys-704 , along with other y series 
  consistent with fragmentation within the glutathione adduct 
  ( fig .
the b 
  series ions , notably b14 , contained no glutathione demonstrating the 
  glutathionylation of the terminal cys-704 residue .
the fragmentation patterns 
  for the nem - alkylated peptides containing cys-531 and cys-704 ( 1591.8 and 
  1637.7 da , respectively ) from purified complex i were similar to those seen 
  for the glutathionylated peptides ( data not shown ) , indicating that 
  glutathionylation does not distort the peptide fragmentation pattern . 
 
therefore , tandem mass spectrometry identifies the modification sites in these 
  peptides and confirms glutathionylation of both cys-531 and cys-704 of the 
  75-kda subunit in diamide - treated bovine heart mitochondria and 
  glutathionylation of cys-531 in diamide - treated rat heart mitochondria .
estimation of the extent of cysteine residue modification in the 
  complex i 75-kda subunit within oxidatively stressed 
  mitochondria  the experiments so far have identified the 
  glutathionylated cys residues within the complex i 75-kda subunit , but the 
  extent of their modification was unclear .
this was estimated by quantifying 
  the loss of the cys residue free thiol , assessed by measuring decreased nem 
  alkylation .
any unmodified cys residues should be equally reactive with nem in 
  diamide - treated and control samples .
in contrast , glutathionylation of a cys 
  residue should lead to decreased recovery of the nem - alkylated peptide that is 
  dtt - sensitive . however , one caveat is that a glutathionylated peptides that 
  broke down during analysis to yield a free thiol that then reacted with nem 
  would not be detected .
figure 4.fragment ion spectra of glutathionylated peptides from complex i 75-kda 
  subunit containing cys-531 and cys-704 .
a , structure of a 
  cys - containing peptide modified by a disulfide link to glutathione .
the sites 
  of fragmentation observed in tandem mass spectrometry experiments are also 
  shown . in subsequent fragment ion spectra
, the asterisk indicates fragment 
  ions containing an intact glutathione molecule , whereas the superscripts 
  , , or   indicate ions 
  resulting from fragmentation at the bond indicated .
b and d , 
  maldi - tof - tof spectra of the glutathionylated peptides obtained from heart 
  mitochondria incubated with diamide as in 
  fig .
1 with complex i 75-kda 
  subunit prepared for mass spectrometry as described in 
  table 2 .
b , fragment 
  ion spectrum of a 1771.8 ion corresponding to a glutathionylated peptide 
  containing cys-531 from bovine complex i. c , fragment spectrum of a 
  1817.8 da ion corresponding to a glutathionylated peptide containing cys-704 
  from bovine complex i. fragment ion spectra of glutathionylated peptides from complex i 75-kda 
  subunit containing cys-531 and cys-704 . a , structure of a 
  cys - containing peptide modified by a disulfide link to glutathione . the sites 
  of fragmentation observed in tandem mass spectrometry experiments are also 
  shown . in subsequent fragment ion spectra , the asterisk indicates fragment 
  ions containing an intact glutathione molecule , whereas the superscripts 
  , , or   indicate ions 
  resulting from fragmentation at the bond indicated .
b and d , 
  maldi - tof - tof spectra of the glutathionylated peptides obtained from heart 
  mitochondria incubated with diamide as in 
  fig .
1 with complex i 75-kda 
  subunit prepared for mass spectrometry as described in 
  table 2 .
b , fragment 
  ion spectrum of a 1771.8 ion corresponding to a glutathionylated peptide 
  containing cys-531 from bovine complex i. c , fragment spectrum of a 
  1817.8 da ion corresponding to a glutathionylated peptide containing cys-704 
  from bovine complex i. to do this , mitochondria were incubated  diamide and then treated 
   dtt before addition of nem
. then complex i was isolated by bn - page ; 
  the 75-kda subunit was separated by nonreducing sds - page , and the 
  nem - alkylated peptides containing the six non - iron - sulfur center cys residues 
  ( 30 , 344 , 531 , 541 , 687 , and 704 ) were analyzed by maldi - tof - ms . to account 
  for differential peptide recovery ,
all peak intensities were normalized 
  relative to a non - cys - containing peptide from the 75-kda subunit ( feaplfnar , 
  [ mh ] 1064.6 da for trypsin digests and dvvllvgtnprf 
  [ mh ] 1329.8 da for asp - n digests ) .
representative experiments for 
  the nem - alkylated peptide containing cys-531 ( 1591.8 da ) and cys-704 ( 1637.7 
  da ) from bovine heart mitochondria are shown in 
  fig .
the peak 
  intensity decreased on diamide treatment ( + diamide ) and was recovered on 
  subsequent incubation with dtt ( + diamide / dtt ) 
  ( fig .
this 
  approach was applied to all six candidate cys residues of the 75-kda subunit 
  in bovine heart mitochondria , and the changes in peak intensity of the 
  relevant nem peptide were determined as a percentage of control mitochondrial 
  incubations ( fig .
diamide led to a statistically significant decrease of 55% in both the 
  cys-531 and cys-704 residues that was reversed by dtt , consistent with 
  extensive glutathionylation of these residues .
in contrast , no significant 
  loss of nem - alkylated peak intensity was observed on diamide treatment for 
  peptides containing cys residues 30 , 344 , 541 , or 687 
  ( fig .
similar 
  analysis of rat heart mitochondria showed that there was an 50% decrease 
  in the intensity of alkylation of the cys-531 residue that was largely 
  reversed by dtt ( data not shown ) . in rat mitochondria ,
cys-704 reacted poorly 
  with nem , so instead the amount of the unalkylated peptide ( 1503.7 da ) was 
  assessed , and its amount decreased by 63% on diamide treatment ( data not 
  shown ) . that these losses of the nem - alkylated peptides were reversed by dtt 
  treatment
indicates that the modification is unlikely to be irreversible 
  oxidation to a sulfinic or sulfonic acid .
furthermore , modification of 
  peptides containing cys-531 and cys-704 to sulfenic , sulfinic , or sulfonic 
  acids was searched for but none was found in mass spectra .
therefore , these 
  data are consistent with extensive glutathionylation of cys-531 and cys-704 in 
  oxidatively stressed bovine heart mitochondria occurring with minimal 
  modification of other cys residues in the 75-kda subunit of complex i. 
  however , the possibility remains that other dtt - reversible modifications may 
  also contribute to the loss of the nem - alkylated peptides seen in 
  fig .
figure 5.quantification of the extent of oxidation of complex i 75-kda subunit 
  cys residues during diamide treatment of mitochondria .
heart mitochondria 
  were incubated  diamide ; some diamide - treated samples were further 
  incubated with dtt , and then all samples were treated with nem .
the peptides 
  containing nem - alkylated cys residues were then analyzed by maldi - tof , and 
  their peak intensities are expressed as a percentage of an appropriate non - cys 
  containing reference peptide peak from the 75-kda subunit digest . a , 
  representative maldi - tof spectra of the nem - alkylated peptides containing 
  cys-531 ( 1591.8 da ) or cys-704 ( 1637.7 da ) from control , diamide - treated , and 
  diamide- and dtt - treated bovine heart mitochondria .
b , effect of 
  treatment of bovine heart mitochondria with diamide or with diamide and dtt on 
  peak intensities of nem - alkylated peptides .
peak intensities are plotted as a 
  percentage of the control incubation ( -diamide ) for that experiment . 
 
data are the means  s.e . of three to four independent experiments , 
  except for the + diamide / dtt experiments , which were done once . , 
  p  0.05 relative to control samples by a paired student 's 
  t test .
quantification of the extent of oxidation of complex i 75-kda subunit 
  cys residues during diamide treatment of mitochondria .
heart mitochondria 
  were incubated  diamide ; some diamide - treated samples were further 
  incubated with dtt , and then all samples were treated with nem .
the peptides 
  containing nem - alkylated cys residues were then analyzed by maldi - tof , and 
  their peak intensities are expressed as a percentage of an appropriate non - cys 
  containing reference peptide peak from the 75-kda subunit digest . a , 
  representative maldi - tof spectra of the nem - alkylated peptides containing 
  cys-531 ( 1591.8 da ) or cys-704 ( 1637.7 da ) from control , diamide - treated , and 
  diamide- and dtt - treated bovine heart mitochondria .
b , effect of 
  treatment of bovine heart mitochondria with diamide or with diamide and dtt on 
  peak intensities of nem - alkylated peptides .
peak intensities are plotted as a 
  percentage of the control incubation ( -diamide ) for that experiment . 
 
data are the means  s.e . of three to four independent experiments , 
  except for the + diamide / dtt experiments , which were done once . , 
  p  0.05 relative to control samples by a paired student 's 
  t test .
glutathionylation of cys residues on the 75-kda of complex i by 
  endogenous oxidative stress within mitochondria  the proximal ros 
  produced by the respiratory chain is superoxide , which rapidly dismutates to 
  h2o2 
  ( 50 ) .
therefore , we next 
  exposed bovine heart mitochondria to oxidative stress in ways designed to 
  mimic the types and amounts of ros encountered by complex i in pathological 
  situations .
this was done by adding h2o2 to 
  mitochondria , or by inducing superoxide production from the respiratory chain 
  at complex i by reverse electron transport or at complex iii using antimycin 
  ( 51 ) .
the glutathionylation of 
  the complex i 75-kda subunit at cys-531 and cys-704 was then assessed as 
  before by maldi - tof - ms .
figure 6.assessment by mass spectrometry of glutathionylation of complex i in 
  mitochondria exposed to endogenous oxidative stress .
bovine heart 
  mitochondria ( 1 mg of protein / ml ) were incubated for 5 min at 37 c . 
 
reverse electron transport ( ret ) incubations were done in 250 
  mm sucrose , 10 mm hepes , 1 mm egta , ph 7.4 , 
  supplemented with 0.01% bovine serum albumin and 10 mm succinate . 
 
antimycin incubations also contained antimycin ( 2.5 m ) and fccp 
  ( 400 nm ) , whereas control incubations were supplemented with fccp 
  ( 400 nm ) alone .
incubations with 25 m 
  h2o2 were done in kcl buffer ( ph 7.2 ) .
complex i was 
  assessed for glutathionylation of the 75-kda subunit cys-531 and cys-704 
  residues by mass spectrometry as described in 
  fig . 1 and 
  table 2 .
a , 
  representative maldi - tof spectra of the glutathionylated peptide containing 
  cys-704 is shown with peak intensities expressed as a percentage of a non - cys 
  containing reference peptide peak from the 75-kda subunit ( feaplfnar , 
  [ mh ] 1064.6 da ) .
b , measurement of the effects of 
  treatment on the peak intensities of the glutathionylated peptides containing 
  the cys-531 and cys-704 residues .
peak intensities are plotted as a percentage 
  of the control incubation and are the means  s.e .
assessment by mass spectrometry of glutathionylation of complex i in 
  mitochondria exposed to endogenous oxidative stress .
bovine heart 
  mitochondria ( 1 mg of protein / ml ) were incubated for 5 min at 37 c . 
 
reverse electron transport ( ret ) incubations were done in 250 
  mm sucrose , 10 mm hepes , 1 mm egta , ph 7.4 , 
  supplemented with 0.01% bovine serum albumin and 10 mm succinate . 
 
antimycin incubations also contained antimycin ( 2.5 m ) and fccp 
  ( 400 nm ) , whereas control incubations were supplemented with fccp 
  ( 400 nm ) alone .
incubations with 25 m 
  h2o2 were done in kcl buffer ( ph 7.2 ) .
complex i was 
  assessed for glutathionylation of the 75-kda subunit cys-531 and cys-704 
  residues by mass spectrometry as described in 
  fig . 1 and 
  table 2 .
a , 
  representative maldi - tof spectra of the glutathionylated peptide containing 
  cys-704 is shown with peak intensities expressed as a percentage of a non - cys 
  containing reference peptide peak from the 75-kda subunit ( feaplfnar , 
  [ mh ] 1064.6 da ) .
b , measurement of the effects of 
  treatment on the peak intensities of the glutathionylated peptides containing 
  the cys-531 and cys-704 residues .
peak intensities are plotted as a percentage 
  of the control incubation and are the means  s.e .
induction of superoxide production by antimycin or by ret , and by direct 
  addition of h2o2 , all led to the glutathionylation of 
  cys-704 ( fig .
6a ) , 
  with only minor changes in cys-531 ( data not shown ) . measurement of these 
  changes over multiple experiments confirmed that these forms of ros led to 
  statistically significant glutathionylation of cys-704 
  ( fig .
therefore , 
  glutathionylation of cys-704 occurs in response to physiologically relevant 
  types and amounts of mitochondrial ros .
earlier we reported that incubation of complex i with 20 
  mm gssg increased superoxide formation during oxidation of nadh , as 
  measured by sod - sensitive reduction of acetylated cytochrome c 
  ( 9 ) .
recently , another group 
  using both spin trapping and acetylated cytochrome c to measure 
  superoxide showed that 13 mm gssg did not increase 
  superoxide production by isolated complex i 
  ( 17 ) .
therefore we reassessed 
  how complex i glutathionylation affected superoxide production by measuring 
  the sod - sensitive clz chemiluminescence by mitochondrial membranes respiring 
  on nadh ( 25 , 
  36 ) .
addition of the complex i 
  inhibitor rotenone increased superoxide formation , indicating that superoxide 
  production from complex i was easily detected by this approach 
  ( fig .
however , 
  incubation with gssg did not increase superoxide production , and further 
  addition of grx2 to accelerate glutathionylation 
  ( fig .
7a ) or varying 
  [ gssg ] from 100 m to 5 mm ( data not shown ) were also 
  ineffective .
10 and data 
  not shown ) indicating that glutathionylation of complex i does not necessarily 
  lead to increased superoxide formation .
possible causes of the discrepancy 
  between these findings and our earlier work may include the high gssg 
  concentrations used , changes in the nadh / nad ratio , and decomposition of 
  complex i during incubations ( data not shown ) .
treatment of isolated mitochondria with tert - butylhydroperoxide 
  increased h2o2 efflux , consistent with increased 
  superoxide formation by glutathionylated complex i 
  ( 9 ) .
however , 
  h2o2 efflux is an indirect measure of superoxide 
  formation as changes in the activity of thiol - dependent mitochondrial 
  h2o2 degradation pathways such as peroxiredoxin iii may 
  also contribute , and glutathione depletion by 1-chloro-2,4-dinitrobenzene has 
  been shown to increase hydrogen peroxide efflux from heart mitochondria 
  ( 52 ) .
therefore , we measured 
  mitochondrial ros production more directly by dihydroethidine oxidation to 
  2-hydroxyethidium or ethidium , which we detected by their fluorescence in the 
  presence of excess dna ( fig . 
 
addition of antimycin to 
  mitochondria respiring on succinate increased dihydroethidine oxidation , 
  consistent with increased superoxide formation by mitochondria .
however , 
  incubation of mitochondria respiring on succinate or on glutamate / malate with 
  various concentrations of diamide for 5 min ( 1 mm , 
  fig .
7b , 10 , 100 , and 
  250 m ; data not shown ) did not increase dihydroethidine 
  oxidation .
mitochondria incubated with clz showed an increase in 
  chemiluminescence on addition of antimycin ( data not shown ) ; however , addition 
  of 0.5 mm diamide to mitochondria respiring on succinate or 
  glutamate / malate did not increase clz chemiluminescence ( data not shown ) .
as 
  diamide treatment of mitochondria under these conditions leads to extensive 
  glutathionylation of complex i , these data , in contrast to our earlier 
  suggestion ( 9 ) , show that 
  complex i glutathionylation within intact mitochondria does not correlate with 
  increased superoxide formation .
a , 
  superoxide formation by bovine mitochondrial membranes was determined by 
  measuring clz chemiluminescence .
mitochondrial membranes were incubated with 1 
  mm nadh , 500 m gssg , 4 g / ml rotenone , 1 unit / ml 
  grx2 , or 100 units / ml cuzn - sod as indicated .
b , oxidation of dihydroethidine 
  by rat heart mitochondria incubated with diamide ( 1 mm ) or 
  antimycin ( 2.5 m ) .
c , rat heart mitochondria ( 0.5 mg protein / ml ) were incubated in 120 
  mm kcl , 10 mm hepes , 1 mm egta , ph 7.5 , at 37 
  c for 5 min with 10 mm glutamate / malate with the indicated 
  concentrations of diamide .
d , rat heart 
  mitochondria were incubated with 0.5 mm diamide for 5 min as in 
  c and pelleted by centrifugation , and then the mitochondria were 
  broken by three cycles of freeze / thawing and incubated with dtt ( 1 
  mm ) or -mercaptoethanol ( 5 mm ) for 2 min , when 
  complex i activity was measured .
mitochondria were incubated with 0.5 mm diamide 
  at 37 c for 5 min , pelleted to remove the diamide , and incubated at 25 
  c in kcl buffer ( ph 7.5 ) with 10 mm glutamate / malate for 
  030 min , and then complex i activity was measured .
f and g , effect of 
  diamide on gssg levels in rat heart mitochondria .
the diamide - treated 
  mitochondria were then washed and incubated in kcl buffer ( ph 7.5 ) with 10 
  mm glutamate / malate ( recovery ) for 030 min and 
  assayed for gsh and gssg .
f shows the gsh and gssg contents , and 
  g shows the gsh / gssg ratio .
a , 
  superoxide formation by bovine mitochondrial membranes was determined by 
  measuring clz chemiluminescence .
mitochondrial membranes were incubated with 1 
  mm nadh , 500 m gssg , 4 g / ml rotenone , 1 unit / ml 
  grx2 , or 100 units / ml cuzn - sod as indicated .
b , oxidation of dihydroethidine 
  by rat heart mitochondria incubated with diamide ( 1 mm ) or 
  antimycin ( 2.5 m ) .
c , rat heart mitochondria ( 0.5 mg protein / ml ) were incubated in 120 
  mm kcl , 10 mm hepes , 1 mm egta , ph 7.5 , at 37 
  c for 5 min with 10 mm glutamate / malate with the indicated 
  concentrations of diamide .
d , rat heart 
  mitochondria were incubated with 0.5 mm diamide for 5 min as in 
  c and pelleted by centrifugation , and then the mitochondria were 
  broken by three cycles of freeze / thawing and incubated with dtt ( 1 
  mm ) or -mercaptoethanol ( 5 mm ) for 2 min , when 
  complex i activity was measured .
mitochondria were incubated with 0.5 mm diamide 
  at 37 c for 5 min , pelleted to remove the diamide , and incubated at 25 
  c in kcl buffer ( ph 7.5 ) with 10 mm glutamate / malate for 
  030 min , and then complex i activity was measured .
f and g , effect of 
  diamide on gssg levels in rat heart mitochondria .
the diamide - treated 
  mitochondria were then washed and incubated in kcl buffer ( ph 7.5 ) with 10 
  mm glutamate / malate ( recovery ) for 030 min and 
  assayed for gsh and gssg .
f shows the gsh and gssg contents , and 
  g shows the gsh / gssg ratio .
effect of mitochondrial exposure to diamide on complex i 
  activity  exposure of mitochondrial membranes to gssg , or to the 
  disulfides cystine and -hydroxyethylenedisulfide , leads to the loss of 
  complex i activity ( 9 , 
  10 ) .
interestingly , this loss 
  of activity is not ameliorated by treatment with thiol reductants , even if 
  complex i glutathionylation is reversed 
  ( 10 ) . to see if there was a 
  similar relationship between complex i activity and exposure to an oxidized 
  glutathione pool within mitochondria , we exposed rat heart mitochondria to 
  various concentrations of diamide for 5 min 
  ( fig .
this led to 
  extensive loss of complex i and of complex ii / iii activity 
  ( fig .
exposure of 
  isolated mitochondria to diamide ( 0.52 mm ) also decreased 
  both coupled and phosphorylating respiration on succinate or glutamate / malate 
  ( data not shown ) .
these data are consistent with diamide exposure leading to 
  oxidation of the mitochondrial glutathione pool with effects on a range of 
  mitochondrial processes . to see
if the loss of complex i activity on diamide treatment was readily 
  reversible , we incubated mitochondria with diamide , lysed the mitochondria , 
  and exposed them to the thiol reductants dtt , -mercaptoethanol 
  ( fig .
7d ) or of complex ii / iii ( data not shown ) , even though 
  these treatments readily removed glutathione residues from complex i ( figs . 
  1 and 
  3 ) ( data not shown ) . to see if 
  restoration of the glutathione pool within intact mitochondria could result in 
  reactivation of complex i , we first exposed mitochondria to diamide and then 
  washed them to remove the oxidant ( fig . 
 
subsequent incubation of these mitochondria for up 
  to 1 h did not lead to restoration of complex i activity , even though the 
  mitochondrial glutathione pool was partially restored 
  ( fig . 7 , f and 
  g ) .
therefore within intact mitochondria , exposure to the thiol oxidant diamide 
  leads to the rapid inactivation of complex i ; however , subsequent exposure to 
  thiol reductants does not restore complex i activity .
this is similar to the 
  situation for complex i exposed to gssg in membranes and indicates that 
  exposure to an oxidized glutathione pool rapidly leads to the inactivation of 
  complex i , but there is not a simple correlation between complex i 
  glutathionylation and its activity .
even so , complex i glutathionylation may 
  affect its function by other mechanisms , such as by the recruitment of binding 
  partners .
location of glutathionylated cys residues on complex i  of 
  the non - iron - sulfur cys residues , cys-30 and cys-344 were not glutathionylated 
  in isolated complex i ; cys residues 531 , 541 , 687 , and 704 were 
  glutathionylated in isolated complex i , whereas only cys-531 and cys-704 were 
  glutathionylated in intact mitochondria .
the six non - iron - sulfur center cys 
  residues in the bovine and rat complex i 75-kda subunit are conserved in 
  mammals , g. gallus , and x. laevis , and all except cys-541 
  are conserved in d. rerio . however only cys-30 and cys-687 are 
  conserved more widely ; cys-30 is conserved in r. americana , c. elegans , n. 
  crassa , a. thaliana , and d. discoideum ; cys-687 is conserved in 
  d. melanogaster , n. crassa , a. thaliana , and a. castellanii , 
  but not in c. elegans or d. discoideum . the location and 
  environment of these cys residues within complex
the structure of mammalian complex i is 
  not known , but that of the hydrophilic arm of complex i from t. 
  thermophilus has been determined 
  ( 4 , 
  53 ) ( protein data bank 2fug ) . 
 
comparisons of the sequence alignments between orthologous subunits of bovine 
  and thermus complex i , and a model of the bovine complex i built by 
  comparative modeling with the t. thermophilus structure , were used to 
  identify the location of the six non - iron - sulfur center cys residues 
  ( fig .
the model of the 
  structure of bovine complex i derived from the t. thermophilus 
  structure is shown in fig . 
 
the 
  six non - iron - sulfur center cys residues shown in 
  fig .
cysteine residues 344 , 531 , 541 , 687 , and 704 are close to 
  each other and are surrounded by a large red oval in 
  fig .
the iron - sulfur centers 
  are indicated as yellow and gray spheres in 
  fig . 8a .
the flavin 
  mononucleotide where nadh is oxidized is indicated , and the probable locations 
  of the mitochondrial inner membrane and the coenzyme q - binding sites are 
  indicated .
figure 8.model of the bovine complex i 75-kda subunit . a model comprising 
  subunits of the bovine complex i that are orthologous to t. 
  thermophilus subunits was built by using comparative modeling . 
  a , structure of the bovine complex i. the 75-kda subunit is colored 
  dark gray , and the six noniron - sulfur cys residues are shown with a 
  yellow stick representation and outlined with a red oval 
  ( cys-344 , cys-531 , cys-541 , cys-687 , and cys-704 ) and a red circle 
  ( cys-30 ) .
the remaining subunits are shown in light gray , and the 
  iron - sulfur centers are shown as yellow and gray spheres ; 
  the location of the flavin mononucleotide ( fmn ) , which accepts 
  electrons from nadh , is indicated by an arrow , the approximate 
  locations of the membrane surface and the coenzyme q are shown as a dashed 
  blue line and a q , respectively .
b , close - up of the 
  complex i 75-kda subunit showing the location of five of the non - iron - sulfur 
  cys residues .
the side chains of the four non - iron - sulfur cys residues that are on 
  the surface of the subunit are colored yellow .
d , local environment 
  of the cys-531 residue in complex i 75-kda subunit showing the close proximity 
  of the two basic residues , arg-534 and lys-516 .
model of the bovine complex i 75-kda subunit . a model comprising 
  subunits of the bovine complex i that are orthologous to t. 
  thermophilus subunits was built by using comparative modeling . 
  a , structure of the bovine complex i. the 75-kda subunit is colored 
  dark gray , and the six noniron - sulfur cys residues are shown with a 
  yellow stick representation and outlined with a red oval 
  ( cys-344 , cys-531 , cys-541 , cys-687 , and cys-704 ) and a red circle 
  ( cys-30 ) .
the remaining subunits are shown in light gray , and the 
  iron - sulfur centers are shown as yellow and gray spheres ; 
  the location of the flavin mononucleotide ( fmn ) , which accepts 
  electrons from nadh , is indicated by an arrow , the approximate 
  locations of the membrane surface and the coenzyme q are shown as a dashed 
  blue line and a q , respectively .
b , close - up of the 
  complex i 75-kda subunit showing the location of five of the non - iron - sulfur 
  cys residues .
the side chains of the four non - iron - sulfur cys residues that are on 
  the surface of the subunit are colored yellow .
d , local environment 
  of the cys-531 residue in complex i 75-kda subunit showing the close proximity 
  of the two basic residues , arg-534 and lys-516 . in this structural model
the bovine residue cys-30 aligned to val-23 in the 
  thermus nqo3 subunit . however , val-23 is in an -helix and does 
  not have a solvent - accessible surface because it is masked by a -sheet . 
 
the side chain is very near the surface of the protein , but it is obstructed 
  from the solvent by the loop region of the neighboring -strand .
the 
  bovine residue cys-531 corresponds to an insertion in a short -helical 
  region of the thermus subunit ( pro-572 to leu-577 ) which is conserved 
  in mammals , g. gallus , x. laevis , and d. rerio .
the 
  -helix is located on the surface of the subunit , and thus cys-531 is 
  likely to be solvent - accessible .
the bovine residue cys-541 is found in the 
  middle of the parallel -sheet containing cys-344 , and the side chain of 
  cys-541 is solvent - accessible .
bovine cys-687 aligns with arg-750 in the 
  thermus subunit , which is in a loop region following the last 
  -turn in the subunit .
the side chain of the residue is partially exposed 
  to the solvent ; however , two loop regions lie on either side of it and may 
  hinder access .
the bovine residue cys-704 is at the c terminus of the protein 
  sequence and corresponds to the end of the sequence ( ala-767 ) in the 
  thermus structure .
as bovine complex i contains 30 more subunits than 
  that of t. thermophilus , it is possible that cys residues that are 
  apparently surface - accessible in our model of the bovine 75-kda subunit may 
  actually be occluded by a subunit not present in t. thermophilus . 
  allowing for this caveat
, the modeling of the 75-kda subunit suggests that 
  cys-30 and cys-344 were not glutathionylated because they were not exposed to 
  the glutathione pool in solution .
in contrast , the four cys residues 
  glutathionylated in the purified bovine 75-kda subunit ( cys-531 , -541 , -687 , 
  and -704 ) are solvent - accessible and thus potentially accessible to 
  glutathionylation . in fig . 
  8b
, the 75-kda subunit is enlarged , and 
  underlining shows with the location of the four non - iron - sulfur 
  center cys residues that are solvent - exposed , as well as cys-344 which is not 
  solvent - exposed .
this is extended in fig . 
  8c , which shows the 75-kda subunit as a space - filling 
  model with the four solvent - exposed cys residues colored yellow .
the modeling data are consistent with the glutathionylation of 
  solvent - exposed cys residues 531 , 541 , 687 , and 704 in purified complex i. 
  however , it was unclear why there was preferential glutathionylation of 
  cys-531 and cys-704 in mitochondria .
the local environment of cys residues can 
  alter their reactivity by decreasing the pka values 
  through the proximity of positively charged amino acid residues , increasing 
  the proportion in the reactive thiolate form 
  ( 54 , 
  55 ) .
we used the model of the 
  bovine 75-kda complex i subunit to see if there were any positive amino acid 
  residues in the vicinity of the four solvent accessible cys residues .
cys-531 
  has a neighboring arg-557 and lys-539 within 5  of the residue 
  ( fig .
cys-541 has 
  lys-562 in close proximity , although its side chain is on the opposite face of 
  the -sheet , whereas arg-325 is 8  away .
cys-687 has arg-655 
  within 5  , and although lys-709 is close its side chain is probably 
  inaccessible .
there are no positively charged residues in the immediate 
  vicinity of cys-704 , although lys-692 is 8  away .
therefore , the 
  proximity of positively charged amino acids close to cys-531 may explain its 
  greater propensity for glutathionylation , although it was unclear why cys-704 
  was also particularly susceptible to glutathionylation .
however , as bovine 
  complex i contains 30 more subunits than that of t. 
  thermophilus , it is possible that the local environment of cys-704 may be 
  affected by a subunit not present in t. thermophilus . to conclude
, modeling the location of the six non - iron - sulfur cys residues 
  in the complex i 75-kda subunit suggests that only four are solvent - accessible 
  and is consistent with their glutathionylation in purified complex i. 
  assessment of the local electrostatic environment of these four cys residues 
  suggests that cys-531 may be the more reactive , hints that cys-541 may be less 
  reactive , but gave little indication of reasons for the different extents of 
  glutathionylation between cys-704 and cys-687 .
conclusions  the glutathionylation of cys-531 and cys-704 is 
  of particular pathological interest because glutathione depletion leads to 
  complex i damage in parkinson disease and other disorders 
  ( 68 , 
  15 , 
  56 ) .
our finding of a 
  significant interaction between cys-531 and cys-704 and the mitochondrial 
  glutathione pool suggests that disruption of this interaction by glutathione 
  depletion may contribute to complex i oxidative damage and dysfunction in 
  pathologies .
reactive nitrogen and oxygen species such as 
  no2 , onoo , 
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  and oh are thought to be particularly important in the 
  oxidative damage to complex i 
  ( 57 , 
  58 ) .
these species react 
  rapidly with thiols as follows : 
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  k 5  10 
  ms 
  ( 59 ) ; onoo , 
  k 700 ms 
  ( 59 ) ; 
  no2 k 35  
  10 ms 
  ( 57 ) ; and 
  oh , k = 24  10 
  ms 
  ( 59 ) .
( in contrast , the direct 
  reaction of thiols with h2o2 or superoxide are slow 
  compared with the reactions of these ros with other antioxidant systems 
  ( 60 , 
  61 ) . )
the fast reaction rates 
  of thiols mean that cys residues on the surface of proteins are likely to 
  react with and inactivate these reactive species near the surface of complex 
  i , thereby preventing damage to other amino acids or prosthetic groups , such 
  as nitration of tyrosine residues by no2 
  ( 57 , 
  58 ) .
these reactions convert 
  the cys thiol to a sulfenic acid or a thiyl radical , preventing further 
  antioxidant protection by the thiol , so the oxidized thiol must be recycled , 
  and this is done by reaction with the mitochondrial glutathione pool and grx2 
  ( 10 , 
  23 ) .
the protein sulfenic acid 
  reacts with gsh to generate a mixed disulfide , whereas the thiyl radical 
  reacts with gsh to generate a disulfide radical anion , which then reacts with 
  o2 to generate superoxide and a mixed disulfide by the winterbourn 
  reaction ( 62 ) , with the 
  superoxide then being detoxified by matrix superoxide dismutase .
the mixed 
  disulfides are returned to a free thiol and gssg by reaction with gsh 
  catalyzed by grx2 ( 24 ) . 
  without the rapid recycling of the sulfenic or thiyl radicals by gsh and grx2 , 
 
the cys thiols would be irreversibly oxidized to sulfinic or sulfonic acids 
  and would be unable to protect complex i from oxidative damage 
  ( 20 , 
  23 , 
  24 ) . this putative antioxidant 
  cycle in the prevention of oxidative damage to complex
deglutathionylation of cys residues by grx2 is an important part of this 
  antioxidant cycle .
there is considerable evidence for a critical antioxidant 
  role for grx2 in mitochondria : decreasing grx2 expression in cells increases 
  oxidative damage and cell death 
  ( 63 , 
  64 ) ; overexpressing grx2 
  protects cells against apoptosis induced by oxidative stress 
  ( 65 ) , and grx2 is maintained 
  in an inactive , iron - sulfur form that is activated by oxidative stress 
  ( 66 ) .
grx2 , but not grx1 , is 
  recycled directly by thioredoxin reductase as well as by gsh , implying that it 
  can remain functional even when the mitochondrial gsh pool is oxidized 
  ( 67 ) .
finally , down - regulation 
  of the cytosolic isoform grx1 renders complex i in mouse brain more 
  susceptible to damage by the parkinsonian neurotoxin 
  1-methyl-4-ophenyl-1,2,3,6-tetrahydropyridine , although how this isoform 
  interacts with complex i is unclear 
  ( 16 ) .
the extensive 
  glutathionylation of cys-531 and cys-704 shows that these cys residues readily 
  interact with the mitochondrial glutathione pool by thiol - disulfide exchange . 
 
glutathionylation can also occur in vivo in the presence of a reduced 
  gsh pool ( 24 ) .
when 
  mitochondria were exposed to h2o2 or to superoxide 
  produced by the respiratory chain , this also led to glutathionylation of 
  complex i , under conditions where there is negligible oxidation of the 
  glutathione pool ( 51 ) . under 
  these conditions glutathionylation
probably occurs following ferrous iron 
  released from fes centers by superoxide reacting with 
  h2o2 to form oh by the fenton 
  reaction , leading to oxidation of complex i thiols to thiyl radicals that are 
  then converted to a glutathionylated cys residue and then to a free thiol by 
  glutathione .
therefore , in these experiments we have probably trapped a small 
  amount of glutathionylated cys residues as it is being recycled by the gsh 
  pool after having detoxified a oh radical 
  ( fig .
this contrasts with 
  diamide treatment where the oxidized glutathione pool renders the cys residues 
  predominantly glutathionylated and no longer protective , perhaps contributing 
  to the nonreversible damage to complex i that accumulates , whereas under 
  milder oxidative stress damage to complex i was reversible by thiol reductants 
  ( 68 , 
  15 , 
  56 ) .
it may be that the other 
  two exposed cys residues , cys-541 and cys-687 , also contribute to this 
  antioxidant cycle but that their glutathionylation is too transient to be 
  measured under these conditions .
figure 9.possible mechanism of protection against oxidative damage to complex i 
  by exposed surface cys residues .
an exposed thiol on the surface of 
  complex i can react rapidly with a range of species such as 
  oh and no2 to generate a 
  thiyl radical , or with onoo or h2o2 to 
  generate a sulfenic acid .
these oxidized thiols can then be oxidized further 
  by o2 to sulfinic or sulfonic acids ( -son ) , 
  thereby preventing the thiols from protecting complex i from oxidative damage 
  to essential amino acid residues ( x ) , leading to an irreversibly 
  damaged complex i. alternatively , the sulfenic acid can react with gsh to form 
  a mixed disulfide with gsh , or the thiyl radical can react with gsh to form a 
  disulfide radical anion that loses an electron to o2 to form 
  superoxide 
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the mixed disulfides then react rapidly with gsh catalyzed by grx2 to give 
  gssg , and the protein thiol is regenerated .
possible mechanism of protection against oxidative damage to complex i 
  by exposed surface cys residues .
an exposed thiol on the surface of 
  complex i can react rapidly with a range of species such as 
  oh and no2 to generate a 
  thiyl radical , or with onoo or h2o2 to 
  generate a sulfenic acid .
these oxidized thiols can then be oxidized further 
  by o2 to sulfinic or sulfonic acids ( -son ) , 
  thereby preventing the thiols from protecting complex i from oxidative damage 
  to essential amino acid residues ( x ) , leading to an irreversibly 
  damaged complex i. alternatively , the sulfenic acid can react with gsh to form 
  a mixed disulfide with gsh , or the thiyl radical can react with gsh to form a 
  disulfide radical anion that loses an electron to o2 to form 
  superoxide 
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the mixed disulfides then react rapidly with gsh catalyzed by grx2 to give 
  gssg , and the protein thiol is regenerated .
a further consideration for the protection of complex i by the antioxidant 
  reactions of surface cys residues is their concentration relative to that of 
  gsh close to the surface of complex i. the solvent - exposed surface area of the 
  t. thermophilus nqo3 subunit , the ortholog of the bovine 75-kda 
  subunit , is 20,500  
  ( 53 ) .
the gsh concentration 
  within heart mitochondria is 25 mm , so there are 
  13 gsh molecules within 50  of the 75-kda subunit surface . 
  consequently , the four exposed surface thiols make a significant contribution 
  to local thiol reactivity near the surface of complex i , consistent with a 
  protective role for protein thiols .
reactive thiols are also involved in modulating the complex i 
  active / deactive transition 
  ( 68 ) , and the activity of 
  complex i can be modified by s - nitrosation 
  ( 13 , 
  14 , 
  69 , 
  70 ) .
our analysis suggests 
  that cys-531 may be particularly reactive ; therefore it will be interesting to 
  see whether complex i is s - nitrosated on this residue . as 
  s - nitrosation often leads to glutathionylation 
  ( 71 ) , s - nitrosation 
  and glutathionylation may turn out to be aspects of a related system to 
  modulate and protect complex i activity 
  ( 72 ) .
although more work is required to assess the importance of surface cys 
  residues in protecting complex i against oxidative damage , our results suggest 
  that the interplay of thiols on the surface of complex i with the glutathione 
  pool and grx2 within mitochondria may be an important mode of preventing 
  oxidative damage to complex i.
 complex i has reactive thiols on its surface that interact with the 
  mitochondrial glutathione pool and are implicated in oxidative damage in many 
  pathologies . however , the cys residues and the thiol modifications involved 
  are not known . here 
 we investigate complex i thiol modification within 
  oxidatively stressed mammalian mitochondria , containing physiological levels 
  of glutathione and glutaredoxin 2 . 
 in mitochondria incubated with the thiol 
  oxidant diamide , complex i is only glutathionylated on the 75-kda subunit . 
 of 
  the 17 cys residues on the 75-kda subunit , 6 are not involved in iron - sulfur 
  centers , making them plausible candidates for glutathionylation . 
 mass 
  spectrometry of complex i from oxidatively stressed bovine heart mitochondria 
  showed that only cys-531 and cys-704 were glutathionylated . 
 the other four 
  non - iron - sulfur center cys residues remained as free thiols . 
 complex i 
  glutathionylation also occurred in response to relatively mild oxidative 
  stress caused by increased superoxide production from the respiratory chain . 
  although complex i glutathionylation within oxidatively stressed mitochondria 
  correlated with loss of activity , it did not increase superoxide formation , 
  and reversal of glutathionylation did not restore complex i activity . 
  
 comparison with the known structure of the 75-kda ortholog nqo3 from 
  thermus thermophilus complex i suggested that cys-531 and cys-704 are 
  on the surface of mammalian complex i , exposed to the mitochondrial 
  glutathione pool . 
 these findings suggest that cys-531 and cys-704 may be 
  important in preventing oxidative damage to complex i by reacting with free 
  radicals and other damaging species , with subsequent glutathionylation 
  recycling the thiyl radicals and sulfenic acids formed on the cys residues 
  back to free thiols . 
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Supplementary Material