An electrogenic redox loop in sulfate reduction reveals a likely widespread mechanism of energy conservation

The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H+/e− ratio of 1. Although the complex does not act as a H+-pump, QrcD may include a conserved proton channel leading from the N-side to the P-side menaquinone pocket. Our work provides evidence of how energy is conserved during dissimilatory sulfate reduction, and suggests mechanisms behind the functions of related bacterial respiratory complexes in other bioenergetic contexts

Monte Carlo simulations of proton pumps: On the working principles of the biological valve that controls proton pumping in cytochrome c oxidase

Gaining a detailed understanding of the proton-pumping process in cytochrome c oxidase (COX) is one of the challenges of modern biophysics. Recent mutation experiments have highlighted this challenge by showing that a single mutation (the N139D mutation) blocks the overall pumping while continuing to channel protons to the binuclear center without inhibiting the oxidase activity. Rationalizing this result has been a major problem because the mutation is quite far from E286, which is believed to serve as the branching point for the proton transport in the pumping process. In the absence of a reasonable explanation for this important observation, we have developed a Monte Carlo simulation method that can convert mutation and structural information to pathways for proton translocation and simulate the pumping process in COX on a millisecond and even subsecond time scale. This tool allows us to reproduce and propose a possible explanation to the effect of the N139D mutation and to offer a consistent model for the origin of the “valve effect” in COX, which is crucial for maintaining uphill proton pumping. Furthermore, obtaining the first structure-based simulation of proton pumping in COX, or in any other protein, indicates that our approach should provide a powerful tool for verification of mechanistic hypotheses about the action of proton transport proteins

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O(2)-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located ≈30 Å from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK(a) of an internal proton donor, Glu-286, from ≈9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pK(a) to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P → F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process

Activationless electron transfer through the hydrophobic core of cytochrome c oxidase

Electron transfer (ET) within proteins occurs by means of chains of redox intermediates that favor directional and efficient electron delivery to an acceptor. Individual ET steps are energetically characterized by the electronic coupling V, driving force ΔG, and reorganization energy λ. λ reflects the nuclear rearrangement of the redox partners and their environment associated with the reactions; λ ≈ 700–1,100 meV (1 eV = 1.602 × 10(-19) J) has been considered as a typical value for intraprotein ET. In nonphotosynthetic systems, functional ET is difficult to assess directly. However, using femtosecond flash photolysis of the CO-poised membrane protein cytochrome c oxidase, the intrinsic rate constant of the low-ΔG electron injection from heme a into the heme a(3)-Cu(B) active site was recently established at (1.4 ns)(-1). Here, we determine the temperature dependence of both the rate constant and ΔG of this reaction and establish that this reaction is activationless. Using a quantum mechanical form of nonadiabatic ET theory and common assumptions for the coupled vibrational modes, we deduce that λ is <200 meV. It is demonstrated that the previously accepted value of 760 meV actually originates from the temperature dependence of Cu(B)–CO bond breaking. We discuss that low-ΔG, low-λ reactions are common for efficiently channeling electrons through chains that are buried inside membrane proteins