Fig. 2.

a The mechanism of H⁺_(P) uptake and ejection from the binuclear center (BNC), upon entry of the 1st electron in the metal reduction phase. In the oxidized enzyme, the charge of Cu_B ⁽²⁺⁾ is neutralized by two counter ions, the anionic ligands, ⁽¹⁻⁾OH and ⁽¹⁻⁾H334 (1). Reduction of Cu_B ⁽²⁺⁾ to CuB⁽¹⁺⁾ by the 1st electron causes a localized charge imbalance on CuB so that one of its ionically bonded ligands must protonate (2). Proton gating of the D- and K-channels regulates proton movements into the BNC. The K-Gate is closed, so that electron entry into the BNC must be accompanied by ^(1-)H334 protonation, by H⁺_(P) from the H⁺-PWC. Formation of HH334 triggers a change in the configuration of His-Tyr and opens the K-gate (3). This opening of the K-gate allows a H⁺_(SK) to travel from H⁺-K362 into the BNC, protonating Cu_B ⁽¹⁺⁾-OH⁽¹⁻⁾ and forming Cu_B⁽¹⁺⁾… OH₂, and also closing the K-gate. The formation of CuB⁽¹⁺⁾…OH₂ has deprived CuB⁽¹⁺⁾ of an anionic ligand. The charge balance of CuB⁽¹⁺⁾ is restored by HH334 deprotonation, restoring the ionic ligand ^(1-)H334. The H⁺_(P) is ejected from the BNC and is transferred to W1a₃ (4). The subsequent reprotonation of K362 from the inner bulk generates an electrostatic potential that forces the H⁺_(P) from H⁺-W1a₃ into the ‘proton hole’ previously generated in the vestibule, ^(1-)HO-W1_Mg. Before entry of the 2nd electron into the BNC there is a reorganization reaction, whereby an electron and proton are transferred from Cu_B to heme a₃: ^(1-)HO-a₃⁽³⁺⁾ + ⁽¹⁻⁾H334-Cu_B⁽¹⁺⁾…OH₂ → H₂O…a₃ ⁽²⁺⁾ + ^(1-)H334-Cu_B ⁽²⁺⁾-OH^(1-), the pumping form of CuB in the metal reduction phase (1). The input of the 2nd electron then proceeds as the 1st. Fig. 2 b shows the mechanism of H⁺_(P) uptake and ejection from the BNC upon entry of the 3rd electron in the oxygen reduction phase, initiated by the reaction of the two-electron reduced BNC with molecular oxygen. This generates a ferryl (a₃ ⁽⁴⁺⁾=O⁽²⁻⁾), a radical (His-•Tyr) and CuB in the form ^(1-)H334–Cu_B ⁽²⁺⁾-OH⁽¹⁻⁾ (1). Reduction of His-•Tyr to ⁽¹⁻⁾His-Tyr by the 3rd electron causes a localized charge imbalance on CuB so that one of its ionically bonded ligands must protonate (2). Gating of the D- and K-channels ensures that electron entry is accompanied by the protonation of ⁽¹⁻⁾H334 with a H⁺_(P) from the H⁺-PWC of the D-channel. Formation of HH334 triggers a change in the configuration of ⁽¹⁻⁾His-Tyr and opens up a pathway connecting HE286 to ⁽¹⁻⁾His-Tyr, allowing a H⁺_(SD) to protonate ⁽¹⁻⁾His-Tyr (3). The protonation of ⁽¹⁻⁾His-Tyr deprives Cu_B⁽²⁺⁾ of an anionic ligand, and so HH334 undergoes deprotonation so as to restore the charge balance of Cu_B ⁽²⁺⁾. The deprotonation of HH334 results in H⁺_(P) ejection from the BNC, transferring it to W1a₃ (4). The subsequent reprotonation of ⁽¹⁻⁾E286 by a H⁺_(SD) from the inner bulk generates an electrostatic potential that forces the H⁺_(P) from H⁺-W1a₃ into ^(1-)HO-W1_Mg. Before entry of the 4^th electron into the BNC there is a reorganization reaction, whereby an electron and proton are transferred from His-Tyr to heme a₃: a₃⁽⁴⁺⁾=O⁽²⁻⁾ +(His-Tyr)Cu_B⁽²⁺⁾→ ⁽¹⁻⁾HO-a₃⁽³⁺⁾ + (His-•Tyr)Cu_B⁽²⁺⁾, the pumping form of Cu_B in the oxygen reduction phase (1). The input of the 4th electron then proceeds as the 3rd.