Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b and heme b. It is concluded that 1) O binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm] → E107 → E99 (heme b) → E445 (heme d) → oxygenated heme d

Evolution of the cytochrome-bd type oxygen reductase superfamily and the function of cydAA in Archaea

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify 3 families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA’ cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA’ from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA’ is the first isoform of cytbd containing only b-type hemes shown to be active when isolated, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d

Transmembrane proton translocation by cytochrome c oxidase

AbstractRespiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O2-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O2 reduction and how these reactions might be energetically coupled to proton pumping across the membrane

Structures of the intermediates in the catalytic cycle of mitochondrial cytochrome c oxidase

Cytochrome c oxidase is the terminal complex of the respiratory chains in the mitochondria of nearly all eu-karyotes. It catalyzes the reduction of molecular O-2 to water using electrons from the respiratory chain, delivered via cytochrome c on the external surface of the inner mitochondrial membrane. The protons required for water formation are taken from the matrix side of the membrane, making catalysis vectorial. This vectorial feature is further enhanced by the fact that the redox catalysis is coupled to the translocation of protons from the inside to the outside of the inner mitochondrial membrane. We are dealing with a molecular machine that converts redox free energy into a protonmotive force (pmf). Here, we review the current extensive knowledge of the structural changes in the active heme-copper site that accompany catalysis, based on a large variety of time-resolved spectroscopic experiments, X-ray and cryoEM structures, and advanced computational chemistry.Peer reviewe

Specific inhibition of proton pumping by the T315V mutation in the K channel of cytochrome ba3 from Thermus thermophilus

Cytochrome ba3 from Thermus thermophilus belongs to the B family of heme‑copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme‑copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme‑copper centre (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba3. The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 μs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle.Peer reviewe

A positive feedback-based gene circuit to increase the production of a membrane protein

<p>Abstract</p> <p>Background</p> <p>Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses.</p> <p>Results</p> <p>In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome <it>bd </it>quinol oxidase in <it>E. coli</it>, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the <it>lux </it>operon activator LuxR from <it>V. fischeri </it>in an autoregulatory, positive feedback configuration.</p> <p>Conclusions</p> <p>Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the <it>bd </it>oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.</p

Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase

AbstractWe have investigated the electron–proton coupling during the peroxy (PR) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of CuA is increased by ∼100 mV, which slows electron transfer to the binuclear centre during the F→O transition by a factor of ∼200. In ED(I-286) proton uptake during PR→F was slowed by a factor of ∼5, which indicates that E(I-286) is the proton donor to PR. In addition, in the mutant enzyme the F→O transition rate displayed a deuterium isotope effect of ∼2.5 as compared with ∼7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Ådelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F→O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of ∼8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled