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Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a protonconducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H 3 O ؉ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O 2 bound to heme a 3 . To block backward proton movement, the water channel remains closed after O 2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O 2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg 2؉ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu 198 , which bridges the Mg 2؉ and Cu A (the initial electron acceptor from cytochrome c) sites, suggest that the Cu A -Glu 198 -Mg 2؉ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg 2؉ -containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.Cytochrome c oxidase (CcO) 3 reduces molecular oxygen (O 2 ) in a reaction coupled with a proton pumping process. After binding of O 2 to the O 2 reduction site (which includes two redox-active metal sites, heme a 3 and Cu B ), four electron equivalents are sequentially donated from cytochrome c via two additional redox active metal sites, Cu A and heme a. Each of the four electron transfers is coupled to the pumping of a single proton equivalent (1, 2).High resolution x-ray structural studies together with mutational analyses for bovine CcO show a possible proton pumping pathway, known as the H-pathway, which includes a hydrogen bond network and a water channel functioning in tandem (1, 2). The water channel provides access of water molecules (or H 3 O ϩ ions) inside the mitochondrial inner membrane (the N-side) to one end of the hydrogen bond network that extends to the outside of the mitochondrial inner membrane (the P-side). The hydrogen bond network interacts tightly with heme a by forming two hydrogen bonds between the formyl group of heme a and Arg 38 in the hydrogen bond network and between the A-ring propionate of heme a and a fixed water molecule in the hydrogen bond network (1, 2). The net positive charge increase in heme a, which occurs upon electron donation from heme a to the O 2 reduction site to be delocalized to the formyl and propionate gro...

Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, we determined the structure of the mammalian Cyt.c–CcO complex at 2.0‐Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter‐molecular span, one of which lies between the other two layers without significant direct interaction with either protein. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein–protein interaction at the docking interface represent the first known example of a new class of protein–protein interaction, which we term “soft and specific”. This interaction is likely to contribute to the rapid association/dissociation of the Cyt.c–CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

XFEL and IR analyses suggest that O2 bound at CuB blocks proton backflow for unidirectional H+ transport by water channel closure.

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