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...
Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.
Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.
The X-ray structure of cyanide-bound bovine heart cytochromecoxidase in the fully oxidized state was determined at 2.0 Å resolution. The structure reveals that the peroxide that bridges the two metals in the fully oxidized state is replaced by a cyanide ion bound in a nearly symmetric end-on fashion without significantly changing the protein conformation outside the two metal sites.
Fully oxidized cytochrome c oxidase (CcO) under enzymatic turnover is capable of pumping protons, while fully oxidized CcO as isolated is not able to do so upon one-electron reduction. The functional difference is expected to be a consequence of structural differences: [Fe(3+)-OH(-)] under enzymatic turnover versus [Fe(3+)-O(2)(2-)-Cu(2+)] for the as-isolated CcO. However, the electron density for O(2)(2-) is equally assignable to Cl(-). An anomalous dispersion analysis was performed in order to conclusively demonstrate the absence of Cl(-) between the Fe(3+) and Cu(2+). Thus, the peroxide moiety receives electron equivalents from cytochrome c without affecting the oxidation states of the metal sites. The metal-site reduction is coupled to the proton pump.
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