Spectroscopic analysis of moss (Ceratodon purpureus and Physcomitrella patens) recombinant non-symbiotic hemoglobins

Non-symbiotic hemoglobins (nsHbs) are O2-binding proteins widely distributed in land plants, including primitive bryophytes. Little is known about the properties of bryophyte nsHbs. Here, we report the spectroscopic characterization of two moss recombinant nsHbs, CerpurnsHb of Ceratodon purpureus and PhypatnsHb of Physcomitrella patens. Spectra showed that the absorption maxima of the ferrous and ferric forms of recombinant CerpurnsHb are located at 418, 531 and 557 nm and 407, 537, 569 (shoulder) and 632 (shoulder) nm, respectively, and of PhypatnsHb are located at 422, 529 and 557 nm and 407, 531, 571 (shoulder) and 647 (shoulder) nm, respectively. These absorption maxima are similar to those of rice Hb1. Also, the absorption maxima of the oxygenated ferrous form of recombinant CerpurnsHb and PhypatnsHb are located at 412, 541 and 575 nm and 414, 541 and 574 nm, respectively, similar to those of oxygenated rice Hb1 and cowpea leghemoglobin II. This evidence indicates that CerpurnsHb and PhypatnsHb are mostly hexacoordinate and that they bind O2.

Non-symbiotic hemoglobins (nsHbs) are O 2 -binding proteins widely distributed in land plants, from primitive bryophytes to evolved monocots and dicots. 1 Based on O 2 -affinity and sequence similarity nsHbs are classified into type 1 and type 2 (nsHbs-1 and nsHbs-2, respectively). 2,3 The O 2 -affinity of nsHbs-1 is very high because of an extremely low O 2 -dissociation rate constant. [3][4][5] Analyses by X-ray crystallography, site-directed mutagenesis and visible spectroscopy revealed that the extremely low O 2 -dissociation rate constant of nsHbs-1 primarily results from Fe-heme hexacoordination by distal His. 4,6 Absorption spectra of nsHbs-1 are similar to those of other Hbs, however nsHbs-1 exhibit distinctive absorption maxima in the deoxyferrous (Hb 2+ ) form. Specifically, nsHb-1 2+ exhibits peaks at ~526 and ~556 nm, which is characteristic of hexacoordinate Hbs. 4,7 In contrast, pentacoordinate Hbs exhibit a broad peak centered at ~556 nm in their Hb 2+ form. 7,8 Little is known about the biophysical properties of primeval land plant nsHbs (e.g., the postulated ancestors of nsHbs-1 and nsHbs-2 1 ), such as bryophyte nsHbs. To better understand these nsHbs, the structure of a moss (Ceratodon purpureus) nsHb (CerpurnsHb) was modeled. 9 The predicted structure suggested that Fe-heme in this protein is hexacoordinate, however this observation has not been verified experimentally. Here, we report Non-symbiotic hemoglobins (nshbs) are o 2 -binding proteins widely distributed in land plants, including primitive bryophytes. Little is known about the properties of bryophyte nshbs. here, we report the spectroscopic characterization of two moss recombinant nshbs, Cerpurnshb of Ceratodon purpureus and Phypatnshb of Physcomitrella patens. Spectra showed that the absorption maxima of the ferrous and ferric forms of recombinant Cerpurnshb are located at 418, 531 and 557 nm and 407, 537, 569 (shoulder) and 632 (shoulder) nm, respectively, and of Phypatnshb are located at 422, 529 and 557 nm and 407, 531, 571 (shoulder) and 647 (shoulder) nm, respectively. these absorption maxima are similar to those of rice hb1. also, the absorption maxima of the oxygenated ferrous form of recombinant Cerpurnshb and Phypatnshb are located at 412, 541 and 575 nm and 414, 541 and 574 nm, respectively, similar to those of oxygenated rice hb1 and cowpea leghemoglobin II. this evidence indicates that Cerpurnshb and Phypatnshb are mostly hexacoordinate and that they bind o 2 .
DNA sequencing detected no mutations within the inserts of plasmids pET28b::CerpurnsHb and pET28b::PhypatnsHb. Thus, the sequences of recombinant CerpurnsHb and PhypatnsHb were identical to that predicted by the pCR2.1::CerHb and pCRII::B46 plasmids. SDS-PAGE analysis nm, respectively, and in PhypatnsHb at 422, 529 and 557 nm and 407, 531, 571 (shoulder) and 647 (shoulder) nm, respectively, similar to those of rice Hb1. Also, the absorption maxima of the Hb 2+ O 2 form of recombinant CerpurnsHb and PhypatnsHb were located at 412, 541 and 575 nm and 414, 541 and 574 nm, respectively, similar to those of oxygenated rice Hb1 and cowpea LbII ( Table 1). This evidence indicates that CerpurnsHb and PhypatnsHb are hexacoordinate and that they bind O 2 . However, the 531 nm maximum of CerpurnsHb 2+ and 529 nm maximum of PhypatnsHb 2+ are weak compared with the maximum of rice Hb1. 4 This observation suggests that hexacoordination is partial in both CerpuprnsHb 2+ and PyspatnsHb 2+ and that these proteins may exist in a mixture of hexa-and pentacoordinate forms. Thus, it is likely that the O 2 -affinities of CerpurnsHb and PhypatnsHb are higher than those reported for other hexacoordinate land plant nsHbs. An unusual characteristic of the oxygenated CerpurnsHb 2+ and PhypatnsHb 2+ spectra was the existence of absorption peaks at 645 nm ( Fig. 2 and Table 1). These spectra are similar to that from recombinant human histoglobin obtained from E. coli grown in a fermentation apparatus aerated using pure oxygen. 11 This observation suggests that Fe-heme is in the high-spin form in oxygenated CerpurnsHb 2+ and PhypatnsHb 2+ , however the origin of peaks at 645 nm is not known. The results reported here provide knowledge of the spectroscopic properties of bryophyte nsHbs and corroborate the Fe-heme hexacoordination predicted for modeled CerpurnsHb. 9

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Acknowledgments
Authors wish to express their gratitude to Miss Gillian Klucas for English corrections. Work in the authors' laboratory has been funded by SEP-PROMEP (grant no. UAEMor-PTC-01-01/ PTC23) and Consejo Nacional de Ciencia y Tecnología (CoNaCyT grant nos. 25229N and 42873Q), México. C.V.-L. is a postdoctoral fellow supported by CoNaCyT.
showed that recombinant CerpurnsHb and PhypatnsHb of the expected molecular masses (19.6 and 19.9 KD, respectively) were synthesized by E. coli Tuner(DE3)pLacI (Fig. 1). Spectra of recombinant CerpurnsHb and PhypatnsHb were highly similar to those of other Hbs (Fig. 2). The absorption maxima of Hb 2+ and Hb 3+ forms of recombinant CerpurnsHb are located at 418, 531 and 557 nm and 407, 537, 569 (shoulder) and 632 (shoulder)