Inhibition of the Electron Transfer of Plant Cytochrome b<inf>561</inf> by the Modification with Diethylpyrocarbonate: In Search of A Common Mechanism for the Transmembrane Electron Transfer from Ascorbate

Importance of the Conserved Lysine 83 Residue of Zea mays Cytochrome b561 for Ascorbate-Specific Transmembrane Electron Transfer As Revealed by Site-Directed Mutagenesis Studies

“…In addition to these conserved structural features, all cytochromes b 561 aligned in Supporting Information Figure S5 showed strict conservation of five Gly, two Pro, one Gln, and one Lys residues. Further, there were several well-conserved aromatic residues, such as Trp 30 , Phe 49 , Tyr 131 , and Trp 135 (Supporting Information Figure S5). These aromatic residues were proposed previously to have a role(s) for mediating the electron transfer between the two heme prosthetic groups or a structural role to maintain the proper bundling of R-helices in the membranes (10).…”

“…The first target was Lys 83 (corresponding to Lys 85 of bovine adrenal cytochrome b 561 ), that is a highly conserved residue and is locating on the cytosolic loop connecting between helices 2 and 3 and might be spatially very close to the cytosolic heme b prosthetic group (Supporting Information Figure S8) (10) and to the "motif 1" sequence (37). Our site-specific chemical modification studies using diethyl pyrocarbonate (DEPC) on bovine adrenal cytochrome b 561 (12,13) and on WT-Zmb 561 (48,49) showed that this residue had a high reactivity toward the reagent. Further, since such chemical modification caused a distinct inhibition on the fast electron acceptance from AsA, it might have a strong interaction with a negatively charged AsA molecule and is likely to have a very important mechanistic role for the rapid electron acceptance from cytosolic AsA (12,13,47).…”

“…Indeed, the Lys 85 residue is well conserved among animal and plant cytochromes b 561 . Our recent study on WT-Zm b 561 by employing the same strategy indicated that Lys 83 (corresponding to Lys 85 of bovine adrenal cytochrome b 561 ) had similar high reactivity toward DEPC and resulting N-carbethoxylation of the Lys 83 residue was responsible partly for the inhibition of the electron acceptance from AsA , . To clarify the mechanistic role(s) of this positively charged residue during the electron-accepting process, we constructed three mutants, K83A, K83D, and K83E.…”

Electron Transfer Reactions of Candidate Tumor Suppressor 101F6 Protein, a Cytochrome b561 Homologue, with Ascorbate and Monodehydroascorbate Radical

“…The striking inhibitory effect of the DEPC-treatment on the electron accepting ability from AsA is one important aspect of the “concerted proton/electron transfer mechanism” operating in the cytochrome b 561 protein family. ,− Since DEPC reagent specifically attacks a deprotonated nitrogen atom (most likely N δ1 ) of a heme-coordinating imidazole group of cytochrome b 561 , which is the basis of the specific ability of this site for the concerted proton/electron transfer from AsA, almost complete absence of such an inhibitory effect of the DEPC-treatment on human 101F6 protein (Figure S2; Figure ) would suggest that the “concerted proton/electron transfer mechanism” is not operating at the cytosolic heme center of human 101F6 protein. In other words, the N δ1 -carbethoxylation of axial His residue(s) does not occur for human 101F6 protein.…”