Figure 8. Interaction of Ca²⁺ with the exit of proton channel H in bovine heart oxidase.

Structure of the exit part of the H-channel is shown based on the oxidized COX crystal structure, PDB entry 1V54. All the groups shown belong to subunit I (polypeptide A) except for S^II205 from subunit II (polypeptide B). The scheme gives a scenario of proton transfer combined from refs. [44], [45], [49]. Proton trajectory is depicted by red arrows and sequence of the proton transfer steps is indicated by numbers. Oxidation of heme a by the binuclear site brings about a conformational change that unlocks the H-channel below the heme [46] (cf. [50] for an alternative proposal) and pumped proton arrives to the guanidine group of R38 from the N-aqueous phase (step 1) via the input part of the H-channel (cf. Figure 1A ); it travels further via the R38-bound H₂O₃₅₄₅ (that has then to shift closer to Y371), OH group of Y371 and H₂O₃₅₂₈ to finally protonate the backbone carbonyl group of Y440 (steps 2–4). The carbonyl function C = O_Y440 protonated, makes the -NH_S441 acidic (imidic acid) and allows for its facile spontaneous deprotonation by carboxylate of D51 (step 5), converting the S441-Y440 peptide bond to the enol form. The protonated state of D51 is then stabilized by multiple hydrogen bonding to S^II205 and S441. As proposed in [44], the enol form of the S441-Y440 peptide bond returns to the initial keto form (the notorious proton transfer via the peptide bond S441-Y440 [44], [45], [49]) actually in two steps. First, the deprotonated enolic = N_S441 receives proton from the backbone -NH of D442 (step 6, the rate limiting stage of the entire process), which is followed by facile reprotonation of -N^- _D442 by the protonated backbone C-OH_Y440 (step 7) returning the latter to the initial carbonyl state. Upon subsequent reduction of heme a, D51 undergoes reorientation associated with loss of the stabilizing hydrogen bonding to S^II205 and, hence, decreased proton affinity, so that its carboxylic group releases the proton to the P-phase (step 8). Ca²⁺ coordinates to the backbone carbonyl oxygen of S441, and also makes a bond with the carboxylate of D442 via intercalated fixed water molecule (hidden behind the Ca ion in this projection of the structure, cf. H₂O₃₅₄₄ in Figure 1B and see ref. [14]). As discussed in the text, Ca²⁺ is expected to inhibit proton transfer through the exit part of the H-channel and, accordingly, to impede the proton transfer-coupled electron transfer by COX.