Reaction of the NAD(P)H:flavin oxidoreductase from Escherichia coli with NADPH and riboflavin: identification of intermediates

Flavin reductase catalyzes the reduction of free flavins by NAD(P)H. As isolated, Escherichia coli flavin reductase does not contain any flavin prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin substrate in a ternary complex prior to oxidoreduction. The reduction of riboflavin by NADPH catalyzed by flavin reductase has been studied by static and rapid kinetics absorption spectroscopies. Static absorption spectroscopy experiments revealed that, in the presence of riboflavin and reduced pyridine nucleotide, flavin reductase stabilizes, although to a small extent, a charge-transfer complex of NADP+ and reduced riboflavin. In addition, reduction of riboflavin was found to be essentially irreversible. Rapid kinetics absorption spectroscopy studies demonstrated the occurrence of two intermediates with long-wavelength absorption during the catalytic cycle. Such intermediate species exhibit spectroscopic properties similar to those of charge-transfer complexes of oxidized flavin and NAD(P)H, and reduced flavin and NAD(P)+, respectively, which have been identified as intermediates during the reaction of flavoenzymes of the ferredoxin-NADP+ reductase family. Thus, a minimal kinetic scheme for the reaction of flavin reductase with NADPH and riboflavin can be proposed. After formation of the Michaelis complex of flavin reductase with NADPH and riboflavin, a first intermediate, identified as a charge-transfer complex of NADPH and riboflavin, is formed. It is followed by a second charge-transfer intermediate of enzyme-bound NADP+ and reduced riboflavin. The latter decays, yielding the Michaelis complex of flavin reductase with NADP+ and reduced riboflavin, which then dissociates to complete the reaction. These results support the initial hypothesis of a structural similarity between flavin reductase and the enzymes of the ferredoxin-NADP+ reductase family and extend it at a functional level.

Steady-state kinetic studies have shown that Fre functions through an ordered mechanism, with NADPH binding first. This indicates that the protein provides a site which accommodates both the reduced pyridine nucleotide and the flavin substrate in a ternary complex prior to electron transfer (14).
Substrate specificity studies have shown that the isoalloxazine ring of the flavin is the only part of the molecule which is recognized by Fre (14).
Although the overall sequence similarity is low, Fre contains a sequence of four amino acids, R 46 PFS 49 , similar to the R 93 LYS 96 region of spinach FNR. The three-dimensional crystallographic structures of proteins of the FNR family (15)(16)(17)(18)(19)(20)(21)(22) clearly show that these residues, which are highly conserved in all family members, are involved in the interaction between the protein and the flavin isoalloxazine ring. In FNR, Ser96 has been proposed to make a hydrogen bond to the N(5) position of the FAD cofactor (15,16). This residue was found to be essential for activity by site-directed mutagenesis (23). The same approach allowed us to demonstrate that Ser49 of Fre (corresponding to Ser96 of FNR) is indeed involved in flavin binding, and that it is also essential for activity (24). In addition, as in FNR, we have shown that Ser49 of Fre interacts with the N(5) of the isoalloxazine ring of the flavin substrate (24).
On the basis of these observations, the hypothesis can be made that Fre and FNR-family members might share mechanistic features in spite of their different affinities for the flavin, which make Fre a flavin reductase and FNRfamily members flavoproteins.
The mechanism of the reaction of several flavoenzymes of the FNR family with NAD(P)H has been previously studied under pre-steady state conditions in a stopped-flow apparatus (23,(25)(26)(27)(28). In these studies, it was shown that the reduction of the flavin cofactor by NAD(P)H occurs in discrete and NAD(P) + , (iv) decay of the latter to yield the Michaelis complex between reduced enzyme and NAD(P) + product (MC-2), which then dissociates to give the reduced enzyme and free NAD(P) + . The four species are spectrally distinguishable and the absorption properties of CT-1 and CT-2 are well documented for enzymes of this class (26).
The rapid reaction methodology has been thus applied to the reaction catalyzed by Fre. We here report the detection of reaction intermediates, which we identified as enzyme-bound charge-transfer complexes between oxidized riboflavin and NADPH and between reduced riboflavin and NADP + .
These results further support the structural and functional relationship between Fre and the members of the FNR family.  (29) and bovine serum albumin as a standard protein. When applicable, values are shown ± standard deviation.

Materials
Static absorption spectroscopy. Spectra were recorded in quartz cuvettes designed for anaerobic work (30), using a Hewlett-Packard diode array 8452A spectrophotometer interfaced to a Hewlett-Packard 89500A ChemStation.
When necessary, enzyme and reagent solutions were made anaerobic by repeated cycles of evacuation and saturation with nitrogen that had been purified from residual oxygen on passage through a heated BASF catalyst (30). Conditions used during individual experiments are specified in the corresponding figure legends. Photochemical reduction (31) were performed in a bath maintained at 10°C.
Rapid reaction kinetics. Experiments were performed using a Hi-Tech SF-61 stopped-flow spectrophotometer interfaced with a Macintosh IIci computer.
Anaerobiosis in the instrument syringes and plumbing was achieved by flushing them with an anaerobic solution of 10 mM dithionite in 50 mM Tris/HCl, pH 8.5, which was allowed to scavenge oxygen overnight and was substituted by anaerobic 25 mM Hepes/KOH, pH 7.5 containing 1 mM EDTA, 10% glycerol, 2 mM glucose and 10 U/ml glucose oxidase, 2 h prior to the beginning of the experiment. Enzyme solutions were made anaerobic in a tonometer by repeated cycles of evacuation and saturation with oxygen-free nitrogen (30). Substrate solutions were instead made anaerobic by bubbling oxygen-free nitrogen through them for at least 15 min. For each reaction equal volumes (70 l) of enzyme and substrate solutions were rapidly mixed. The KISS program (Kinetic Instruments Inc., USA) was used for data acquisition and analysis. When needed, absorbance spectra of the reaction mixtures of the substrate solutions were recorded using the rapid scanning device of the instrument.

Spectrophotometric titration of riboflavin with Fre.
In order to gather preliminary information on the absorbance properties of various species that may form during the reaction of Fre with NADPH and riboflavin, in a first set of experiments we studied the modification of the absorbance spectrum of Rf induced by addition of Fre. Previous work (32) has shown that binding of Rf to Fre can occur, as monitored by flavin fluorescence quenching. Aliquots of a Fre solution (521 M) were added to a solution of Rf (11.7 M) in 50 mM Hepes/KOH buffer, pH 7.6, 10% glycerol (buffer A), at 10°C. As shown in Figure 1, binding of Rf to Fre caused well defined absorbance changes. The two maxima in the visible region of the free Rf spectrum shifted to longer wavelengths, from 374 to 382 nm, and from 446 to 456 nm, respectively, with a decrease of the extinction coefficient which is more marked for the 446 nm peak. A plot of fractional absorbance changes at 446 nm as a function of the [Fre]/[Rf] ratio is shown in the inset of Figure 1.
The data were fitted to Eq. 1 : At the highest Fre concentration used in the experiment, it was calculated from Eq. 1 that 86.4% Rf was present in complex with Fre. From these data, it was possible to determine the extinction coefficient of the Fre-Rf complex ( Fre-Rf ) at a given wavelength, using Eq. 2 : where : A, absorbance at a given wavelength;  Fre-Rf , extinction coefficient of the Fre-Rf complex at the given wavelength;  Rf , extinction coefficient of free Rf at this wavelength. The extinction coefficients of Fre-Rf were calculated to be 10.7 and 11.5 mM -1 cm -1 at 446 and 456 nm respectively.
In order to study the influence of the pyridine nucleotide on the

The lack of effect of NADP + and AADP on the interaction between Fre and
Rf is in keeping with the fact that NADP + is a poor inhibitor of Fre (K i = 5 mM, 14) and that AADP exerts no inhibition on Fre reaction at concentrations as high as 2 mM (this work).

Fre-catalyzed riboflavin reduction by NADPH.
In order to study the absorbance changes associated with the reaction of Such an absorbance band could be assigned to a charge-transfer complex between reduced Rf and NADP + within Fre active site rather than to a flavin neutral semiquinone species. This conclusion could be reached on the basis of its shape (33)(34)(35)(36)(37), on the fact that it was observed at early stages of the titration when added NADPH had been totally converted to NADP + , and that it persisted until the end of the titration.
In order to confirm this conclusion, the two following experiments were Altogether, these data confirm that the observed long-wavelength absorbance band could be assigned to a charge-transfer complex between enzyme-bound reduced Rf and NADP + . It is likely that the weak intensity of the band reflects the fact that only a small amount of the charge-transfer complex is present as a consequence of its tendency to dissociate to yield the Fre.NADP + .Rf Michaelis complex and the free species, Fre, reduced Rf and NADP + . The latter hypothesis is consistent with the high K i value for NADP + (5 mM, 14).
Kinetics of the Fre-catalyzed reaction between NADPH and Rf.  Figure 4A), but the time-dependence of such changes could not be described by functions corresponding to the sum of exponential processes. Rather, the initial (< 5ms) absorbance decrease at 450 nm was found to be linear with time (data not shown). This observation suggested that, rapidly, and before any significant amount of substrates were used, a state approximating steady-state turnover may be reached in solution.
Indeed, by making the simple minded calculation of the initial reaction velocity of Rf reduction from the initial linear part of the traces observed at various NADPH concentrations, we calculated values that were consistent with an apparent k cat of 67 s -1 and an apparent K m for NADPH of 21 M.
These values are very similar to those estimated from actual initial velocity measurements under steady-state conditions (Table 1).
At all NADPH concentrations tested, absorbance changes in the 520-750 nm range were qualitatively similar to each other. Figure 4A shows a representative trace of the time-course of absorbance changes observed at 550 nm. At all wavelengths between 520 and 750 nm, and at all NADPH concentrations used, only two distinct phases could be observed since a very rapid undetectable primary absorbance increase was completed within the instrument dead-time (1.7 msec) (The initial spectrum has no absorption at wavelengths between 520 and 750 nm). The first detectable phase is characterized by a slight absorbance increase. In the second phase, the absorbance decreases back to the baseline level. The absorbance at 550 nm reached a maximum while Rf was mainly present in the oxidized form, as indicated by the trace at 450 nm, and decayed as Rf got reduced ( Figure 4A).
A good fit of the traces observed between 520 and 750 nm could be obtained using Eq. 3 and the same set of rates, k 1 and k 2 .
where : A, absorbance at time t; A 1 and A 2 , pre-exponential terms which describe the amplitude of each phase; C, absorbance at the end of the reaction.   (Table 1).
At all NADPH concentrations, and at wavelengths between 520 and 800 nm, a rapid initial absorbance increase was now detectable, followed by a further slower increase and a final absorbance decrease back to the baseline level. All traces obtained at various wavelengths and various NADPH concentrations could be fitted with Eq. 4, which describes the sum of three exponential processes ( Figure 4B).
where : A, absorbance; A 1 , A 2 , A 3 , pre-exponential terms which describe the amplitude of each phase; k 1 , k 2 , k 3 , apparent rate constants; C, absorbance at the end of the reaction.
The values calculated for the observed second and third reaction phase ( k 2 , 83  9 s -1 and k 3 , 38  2 s -1 ) were essentially independent from NADPH concentration ( Figure 5). On the contrary, the calculated value of the observed rate associated with the initial rapid reaction phase increased with increasing NADPH concentration. Interestingly, the values of the observed k 1 appeared to show a hyperbolic dependence on NADPH concentration, and a value of 1247  232 s -1 could be extrapolated at infinite NADPH concentration from a double reciprocal plot ( Figure 5). An apparent K m value for NADPH of 199  72 M was determined. However the physical meaning of this parameter cannot be determined at this stage.
The dependence of the observed rate describing the initial reaction phase on NADPH concentration is indeed that expected if this rate reflected that of formation of a charge-transfer complex between Fre-bound Rf and NADPH (analogous to CT-1 of Scheme 1) from the ternary Fre.NADPH.Rf Michaelis complex ( corresponding to MC-1 of Scheme 1). Furthermore, the first reaction phase is significantly faster than the following reaction phases, which, in turn, both appear to be associated with rates greater than that of k cat determined for Fre during steady-state kinetic measurements at 5 °C. Finally, it should be noted that the absorbances of the reaction mixture at 550 nm and 700 nm were maximal while Rf was mainly present in the oxidized form, as indicated by the trace at 450 nm ( Figure 4B).
Altogether, these results can be interpreted in terms of two transient reaction intermediates, whose absorbance at long wavelengths suggests that they may be charge-transfer complexes similar to CT-1 and CT-2 of Scheme 1. On the basis of the observed rates of appearance and decay of such transient species, the proposal that they may be part of the catalytic turnover of Fre can be made. In particular, if the hypothesis were made that the observed rates reflected the rates of conversion of MC-1 into CT-1 (k 1 ), CT-1 into CT-2 (k 2 ) and of CT-2 into MC-2 and free reaction products (k 3 ), it would appear that Fre catalytic turnover is determined by decay of CT-2.

Absorbance spectra of reaction intermediates.
As expected from the results presented above, the long-wavelength absorbing species were found to accumulate to a greater extent during rapid kinetic experiments performed in the presence of higher concentrations of Rf with respect to Fre (37 M Fre, 147 M Rf and 400 M NADPH). From inspection of traces obtained at several wavelengths between 520 and 800 nm, it was clear that two intermediates were formed. The relative amounts of these species reached a maximum at 3 and 75 ms of reaction, respectively. As shown in Figure 6, at early stages of the reaction (up to 3 ms), only 2 % of Rf was reduced and a species with high absorbance at 550 nm and low absorbance at 750 nm was formed. As more Rf was reduced, another species characterized by a lower absorbance at 550 nm, but a relatively higher absorbance at 750 nm accumulated (maximum at 75 ms). Absorbance changes observed at various wavelengths between 520 and 800 nm could be fitted with Eq. 5, which describes the sum of four exponential processes using the same set of rates , k 1 = 1046 s -1 , k 2 = 52 s -1 , k 3 = 31 s -1 , k 4 = 9.6 s -1 (inset of Figure   6). A = A 1 exp(-k 1 *t)+A 2 exp(-k 2 *t)+A 3 exp(-k 3 *t)+A 4 exp(-k 4 *t)+C (5) where : A, absorbance; A 1 , A 2 , A 3 , A 4 , pre-exponential terms, which describe the amplitude of each phase; k 1 , k 2 , k 3 , k 4 , apparent rate constants; C, However, in our case, such intermediate is unstable and decays.
Interestingly, a low amount of (Fre.Rf red .NADP + )* charge-transfer complex was still present at the end of the reaction (Figure 7, spectrum at 1 s) in agreement with the results of the static spectrophotometric experiments ( Figures 2B and 3). In the case of PDR-FeS (26) an extinction coefficient of 3200 M -1 cm -1 at 725 nm was determined for the charge-transfer complex CT-2 formed between reduced flavin and oxidized pyridine nucleotide. Using this value, we can roughly estimate that about 6% of Fre is present as (Fre.Rf red .NADP + )* charge-transfer complex at the end of the reaction ( Figure   7). NADP + , which is an inhibitor of Fre reaction, competitive with respect to NADPH, exhibits a very low affinity for the enzyme, with a K i value of 5 mM (14). At the end of the experiment (Figures 6 and 7 Figure 7. although Fre uses the flavin as a substrate rather than as a prosthetic group, the reduction of Rf by NADPH within the enzyme active center may occur through the same mechanism as that used by enzymes of the FNR family (23,(25)(26)(27)(28).

ACKNOWLEDGMENTS
The stay of Vincent Nivière in Milano to carry out the static and kinetic spectrophotometric work was made possible through a FEBS short-term fellowship. Dr. Bruno Curti is thanked for helpful discussions.