Proton-Coupled Electron Transport in Two Distinct CYBASC Paralogs of : A Comparative Characterization of Highly Conserved Tyrosine and Lysine Residues.

CYBASC proteins are ascorbate (AscH) reducible, diheme -containing integral membrane cytochrome proteins (cyt), which are proposed to be involved in AscH recycling and facilitation of iron absorption. Two distinct CYBASC paralogs from the plant , cyt-A (A-paralog) and cyt-B (B-paralog), have been found to differ in their visible-spectral characteristics and their interaction with AscH and ferric iron chelates. A previously determined crystal structure of the B-paralog provides the first insights into the structural organization of a CYBASC member and implies hydrogen bonding between the substrate AscH and the conserved lysine residues at positions 77 (B-K77) and 81 (B-K81). The function of the highly conserved tyrosine at position 70 (B-Y70) is not obvious in the crystal structure, but its localization indicates the possible involvement in proton-coupled electron transfer. Here we show that B-Y70 plays a major role in the modulation of the oxidation-reduction midpoint potential of the high-potential heme, (), as well as in AscH oxidation. Our results support the involvement of the functionally conserved B-K77 in the stabilization of the dianion Asc. These findings are supported by the crystal structure of the B-paralog, but a comparative biochemical and biophysical characterization of the A- and B-paralogs implied distinct and more complex functions of the corresponding residues A-Y69 and A-K76 in the A-paralog. Our results emphasize the need for a high-resolution crystal structure of the A-paralog to illuminate the differences in functional organization between the two paralogs.