The control of electron flux through cytochrome oxidase.
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ABSTRACT:
SUBMITTER: Murphy MP
PROVIDER: S-EPMC1147883 | biostudies-other | 1987 Apr
REPOSITORIES: biostudies-other
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Project description:The redox states of exogenously added ubiquinone-2 and cytochrome c, and the protonmotive force (delta p) of rat liver mitochondria were measured as the respiration rate was titrated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone. The force ratio delta Eh/delta p across the bc1 complex was close to 1:1 in State 4, indicating an H+/e- stoichiometry of 1:1 for the cytochrome bc1 complex, excluding protons moved by pool ubiquinone. Assuming a constant stoichiometry the rate of electron transport increased linearly with the disequilibrium (delta Eh - delta p) across the complex.
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Project description:We studied the selectivity of a functional model of cytochrome c oxidase's active site that mimics the coordination environment and relative locations of Fe(a3), Cu(B), and Tyr(244). To control electron flux, we covalently attached this model and analogs lacking copper and phenol onto self-assembled monolayer-coated gold electrodes. When the electron transfer rate was made rate limiting, both copper and phenol were required to enhance selective reduction of oxygen to water. This finding supports the hypothesis that, during steady-state turnover, the primary role of these redox centers is to rapidly provide all the electrons needed to reduce oxygen by four electrons, thus preventing the release of toxic partially reduced oxygen species.
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Project description:Cytochrome c (Cytc)1is a cellular life and death decision molecule that regulates cellular energy supply and apoptosis through tissue specific post-translational modifications. Cytc is an electron carrier in the mitochondrial electron transport chain (ETC) and thus central for aerobic energy production. Under conditions of cellular stress, Cytc release from the mitochondria is a committing step for apoptosis, leading to apoptosome formation, caspase activation, and cell death. Recently, Cytc was shown to be a target of cellular signaling pathways that regulate the functions of Cytc by tissue-specific phosphorylations. So far five phosphorylation sites of Cytc have been mapped and functionally characterized, Tyr97, Tyr48, Thr28, Ser47, and Thr58. All five phosphorylations partially inhibit respiration, which we propose results in optimal intermediate mitochondrial membrane potentials and low ROS production under normal conditions. Four of the phosphorylations result in inhibition of the apoptotic functions of Cytc, suggesting a cytoprotective role for phosphorylated Cytc. Interestingly, these phosphorylations are lost during stress conditions such as ischemia. This results in maximal ETC flux during reperfusion, mitochondrial membrane potential hyperpolarization, excessive ROS generation, and apoptosis. We here present a new model proposing that the electron transfer from Cytc to cytochrome c oxidase is the rate-limiting step of the ETC, which is regulated via post-translational modifications of Cytc. This regulation may be dysfunctional in disease conditions such as ischemia-reperfusion injury and neurodegenerative disorders through increased ROS, or cancer, where post-translational modifications on Cytc may provide a mechanism to evade apoptosis.
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Project description: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 DeltaG, and reorganization energy lambda. lambda reflects the nuclear rearrangement of the redox partners and their environment associated with the reactions; lambda approximately 700-1,100 meV (1 eV = 1.602 x 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-DeltaG 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 DeltaG 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 lambda 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-DeltaG, low-lambda reactions are common for efficiently channeling electrons through chains that are buried inside membrane proteins.
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Project description:Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp. str. NT-26 (a member of the Alphaproteobacteria) is a heterotetramer consisting of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Stopped-flow spectroscopy and isothermal titration calorimetry (ITC) have been used to better understand electron transfer through the redox-active centres of the enzyme, which is essential for biosensor development. Results show that oxidation of arsenite at the active site is extremely fast with a rate of >4000s-1 and reduction of the electron acceptor is rate-limiting. An AioB-F108A mutation results in increased activity with the artificial electron acceptor DCPIP and decreased activity with cytochrome c, which in the latter as demonstrated by ITC is not due to an effect on the protein-protein interaction but instead to an effect on electron transfer. These results provide further support that the AioB F108 is important in electron transfer between the Rieske subunit and cytochrome c and its absence in the arsenite oxidases from the Betaproteobacteria may explain the inability of these enzymes to use this electron acceptor.
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Project description:Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain that catalyzes respiratory reduction of dioxygen (O(2)) to water in all eukaryotes and many aerobic bacteria. CcO, and its homologs among the heme-copper oxidases, has an active site composed of an oxygen-binding heme and a copper center in the vicinity, plus another heme group that donates electrons to this site. In most oxidoreduction enzymes, electron transfer (eT) takes place by quantum-mechanical electron tunneling. Here we show by independent molecular dynamics and quantum-chemical methods that the heme-heme eT in CcO differs from the majority of cases in having an exceptionally low reorganization energy. We show that the rate of interheme eT in CcO may nevertheless be predicted by the Moser-Dutton equation if reinterpreted as the average of the eT rates between all individual atoms of the donor and acceptor weighed by the respective packing densities between them. We argue that this modification may be necessary at short donor/acceptor distances comparable to the donor/acceptor radii.
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Project description:Mixed quantum mechanical/molecular mechanics calculations were used to explore the electron pathway of the terminal electron transfer enzyme, cytochrome c oxidase. This enzyme catalyzes the reduction of molecular oxygen to water in a multiple step process. Density functional calculations on the three redox centers allowed for the characterization of the electron transfer mechanism, following the sequence Cu(A)→heme a→heme a(3). This process is largely affected by the presence of positive charges, confirming the possibility of a proton coupled electron transfer. An extensive mapping of all residues involved in the electron transfer, between the Cu(A) center (donor) and the O(2) reduction site heme a(3)-Cu(B) (receptor), was obtained by selectively activating/deactivating different quantum regions. The method employed, called QM/MM e-pathway, allowed the identification of key residues along the possible electron transfer paths, consistent with experimental data. In particular, the role of arginines 481 and 482 appears crucial in the Cu(A)→heme a and in the heme a→heme a(3) electron transfer processes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.
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Project description:Cytochrome c oxidase mediates the final step of electron transfer reactions in the respiratory chain, catalyzing the transfer between cytochrome c and the molecular oxygen and concomitantly pumping protons across the inner mitochondrial membrane. We investigate the electron transfer reactions in cytochrome c oxidase, particularly the control of the effective electronic coupling by the nuclear thermal motion. The effective coupling is calculated using the Green's function technique with an extended Huckel level electronic Hamiltonian, combined with all-atom molecular dynamics of the protein in a native (membrane and solvent) environment. The effective coupling between Cu(A) and heme a is found to be dominated by the pathway that starts from His(B204). The coupling between heme a and heme a(3) is dominated by a through-space jump between the two heme rings rather than by covalent pathways. In the both steps, the effective electronic coupling is robust to the thermal nuclear vibrations, thereby providing fast and efficient electron transfer.
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Project description:Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36?Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.
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