Electron paramagnetic resonance studies of the soluble CuA protein from the cytochrome ba3 of Thermus thermophilus.

The electron paramagnetic resonance (EPR) spectrum of the binuclear CuA center in the water-soluble subunit II fragment from cytochrome ba3 of Thermus thermophilus was recorded at 3.93, 9.45, and 34.03 GHz, and the EPR parameters were determined by computer simulations. The frequency and M1 dependence of the linewidth was discussed in terms of g strain superimposed on a correlation between the A and g values. The g values were found to be gx = 1.996, gy = 2.011, gz = 2.187, and the two Cu ions contribute nearly equally to the hyperfine structure, with magnitude of Ax magnitude of approximately 15 G, magnitude of Ay magnitude = 29 G, and magnitude of Az magnitude of = 28.5 G (65Cu). Theoretical CNDO/S calculations, based on the x-ray structure of the Paracoccus denitrificans enzyme, yield a singly occupied antibonding orbital in which each Cu is pi*-bonded to one S and sigma*-bonded to the other. In contrast to the equal spin distribution suggested by the EPR simulations, the calculated contributions from the Cu ions differ by a factor of 2. However, only small changes in the ligand geometry are needed to reproduce the experimental results

Redox-coupled proton translocation in biological systems: Proton shuttling in cytochrome c oxidase

In the respiratory chain free energy is conserved by linking the chemical reduction of dioxygen to the electrogenic translocation of protons across a membrane. Cytochrome c oxidase (CcO) is one of the sites where this linkage occurs. Although intensively studied, the molecular mechanism of proton pumping by this enzyme remains unknown. Here, we present data from an investigation of a mutant CcO from Rhodobacter sphaeroides [Asn-139 → Asp, ND(I-139)] in which proton pumping is completely uncoupled from the catalytic turnover (i.e., reduction of O(2)). However, in this mutant CcO, the rate by which O(2) is reduced to H(2)O is even slightly higher than that of the wild-type CcO. The data indicate that the disabling of the proton pump is a result of a perturbation of E(I-286), which is located 20 Å from N(I-139) and is an internal proton donor to the catalytic site, located in the membrane-spanning part of CcO. The mutation results in raising the effective pK(a) of E(I-286) by 1.6 pH units. An explanation of how the mutation uncouples catalytic turnover from proton pumping is offered, which suggests a mechanism by which CcO pumps protons

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the “peroxy” state, P(r)) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH ≈ 7.5. The movement of the Lys is proposed to regulate proton transfer by “shutting off” the protonic connectivity through the K-pathway after initiation of the O(2) reduction chemistry. This “shutoff” prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps