Proton transfer in the quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus during reduction of oxygen.

Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246]. In this study, we have investigated the reaction between this qNOR and oxygen. Our results show that, like some cNORs, the G. stearothermophilus qNOR is capable of O(2) reduction with a turnover of ~3electronss(-1) at 40°C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) qNOR reacts with oxygen in a reaction with a time constant of 1.8ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between qNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in qNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Surface-coupled proton exchange of a membrane-bound proton acceptor.

Proton-transfer reactions across and at the surface of biological membranes are central for maintaining the transmembrane proton electrochemical gradients involved in cellular energy conversion. In this study, fluorescence correlation spectroscopy was used to measure the local protonation and deprotonation rates of single pH-sensitive fluorophores conjugated to liposome membranes, and the dependence of these rates on lipid composition and ion concentration. Measurements of proton exchange rates over a wide proton concentration range, using two different pH-sensitive fluorophores with different pK(a)s, revealed two distinct proton exchange regimes. At high pH (> 8), proton association increases rapidly with increasing proton concentrations, presumably because the whole membrane acts as a proton-collecting antenna for the fluorophore. In contrast, at low pH (< 7), the increase in the proton association rate is slower and comparable to that of direct protonation of the fluorophore from the bulk solution. In the latter case, the proton exchange rates of the two fluorophores are indistinguishable, indicating that their protonation rates are determined by the local membrane environment. Measurements on membranes of different surface charge and at different ion concentrations made it possible to determine surface potentials, as well as the distance between the surface and the fluorophore. The results from this study define the conditions under which biological membranes can act as proton-collecting antennae and provide fundamental information on the relation between the membrane surface charge density and the local proton exchange kinetics.

Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway.

Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O(2) to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O(2) reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O(2) reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK(a) of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O(2) reduction, but a decrease in the apparent pK(a) of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.

Deuterium isotope effect of proton pumping in cytochrome c oxidase.

In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron-proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3-->F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3-->O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.

Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase.

Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of O2 to H2O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a "gate," which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molecular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogen/deuterium exchange MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the framework of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation.

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase.

Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations of < or = 250 microM to the outside of the vesicles did not alter the transition rates between intermediates PR (P3)-->F3-->O4. However, proton pumping was impaired specifically during the P3-->F3, but not during the F3-->O4 transition at Zn2+ concentrations of < or = 25 microM. Furthermore, proton pumping during the P3-->F3 transition was typically impaired with the "as isolated" CytcO, which was found to contain Zn2+ ions at microM concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P3-->F3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a KI of approximately 1 microM for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P3-->F3 transition.

The timing of proton migration in membrane-reconstituted cytochrome c oxidase.

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H2O the uptake and release reactions occur with the same rates, in D2O the substrate and pumped protons are taken up first (tau(D) congruent with 200 micros, "peroxy" to "ferryl" transition) followed by a significantly slower proton release to the other side of the membrane (tau(D) congruent with 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation.

A single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter.

Proteins undergo structural fluctuations between nearly isoenergetic substates. Such fluctuations are often intimately linked with the functional properties of proteins. However, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. In this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of O(2) to H(2)O and as a transmembrane proton pump. The segment of the protein where proton translocation is controlled ("gating" region) overlaps with a channel through which O(2) is delivered to the catalytic site. We show that the replacement of an amino acid residue with a small side chain (Gly) by one with a larger side chain (Val), in a narrow part of this channel, completely blocks the O(2) access to the catalytic site and results in formation of a compartment around the site that is impermeable to small gas molecules. Thus, the protein motions cannot counter the blockage introduced by the mutation. These results indicate that the protein motions are restricted in the proton-gating region and that rapid O(2) delivery to the catalytic site requires a gas channel, which is confined within a rigid protein body.

Subunit III of cytochrome c oxidase of Rhodobacter sphaeroides is required to maintain rapid proton uptake through the D pathway at physiologic pH.

The catalytic core of cytochrome c oxidase is composed of three subunits where subunits I and II contain all of the redox-active metal centers and subunit III is a seven transmembrane helix protein that binds to subunit I. The N-terminal region of subunit III is adjacent to D132 of subunit I, the initial proton acceptor of the D pathway that transfers protons from the protein surface to the buried active site approximately 30 A distant. The absence of subunit III only slightly alters the initial steady-state activity of the oxidase at pH 6.5, but activity declines sharply with increasing pH, yielding an apparent pK(a) of 7.2 for steady-state O(2) reduction. When subunit III is present, cytochrome oxidase is more active at higher pH, and the apparent pK(a) of steady-state O(2) reduction is 8.5. Single-turnover experiments show that proton uptake through the D pathway at pH 8 slows from >10000 s(-1) in the presence of subunit III to 350 s(-1) in its absence. At low pH (5.5) the D pathway of the oxidase lacking subunit III regains its capacity for rapid proton uptake. Analysis of the F --> O transition indicates that the apparent pK(a) of the D pathway in the absence of subunit III is 6.8, similar to that of steady-state O(2) reduction (7.2). The pK(a) of D132 itself may decline in the absence of subunit III since its carboxylate group will be more exposed to solvent water. Alternatively, part of a proton antenna for the D pathway may be lost upon removal of subunit III. It is proposed that one role of subunit III in the normal oxidase is to maintain rapid proton uptake through the D pathway at physiologic pH.