Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophoshate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and demethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O2 is served by two major oxidoreductases (oxidases), cytochrome bo3 and cytochrome bd. Terminal oxidases of aerobic respiratory chains of bacteria, which use O2 as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo3 and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo3 and cytochrome bd. The E. coli membrane contains three types of quinones which all have an octaprenyl side chain (C40). It has been proposed that the bo3 oxidase can have two ubiquinone-binding sites with different affinities. The spectral properties of cytochrome bd-II closely resemble those of cydAB-encoded cytochrome bd.

Understanding the photoinduced dynamics of fluorescent proteins is essential for their applications in bioimaging. Despite numerous studies on the ultrafast dynamics, the delayed response of these proteins, which often results in population of kinetically trapped dark states of various origins, is largely unexplored. Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert. This protein shows no excited-state proton transfer during its nanosecond excited-state lifetime; however, its tyrosine-based chromophore undergoes deprotonation coupled to non-radiative electronic relaxation. Such deprotonation causes distinct optical absorption changes in the broad UV-to-NIR spectral range (ca. 300-800 nm); the disappearance of the transient absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of proposed intermediates in the photocycle. We conclude that the non-radiative excited-state decay includes two major branches: internal conversion coupled to intraprotein proton transfer, where a conserved residue E222 serves as the proton acceptor; and ionization induced by two consecutive resonant absorption events, followed by deprotonation of the chromophore radical cation to bulk solvent through a novel water-mediated proton-wire pathway. Our findings open up new perspectives on the dynamics of fluorescent proteins as tracked by its optical transient absorption in the time domain extending up to seconds.

Catalytic mechanisms of reduction of O2 to 2H2O by respiratory terminal oxidases have been extensively investigated. Tri-heme (b558, b595, d) cytochrome bd oxidases presumably utilize a dihemic site composed of high-spin hemes d and b595. We performed a CO photolysis/recombination study of the purified fully reduced cytochrome bd from Escherichia coli. Spectrum of CO photolysis suggests photodissociation of the ligand from heme d and from part of heme b595. This is the first clear evidence of interaction of heme b595 with CO at room temperature. The amount of the heme d-CO species is higher after recombination than before photolysis. In the enzyme population with heme b595 bound to CO, heme d remains unliganded, hence the dihemic O2-reducing pocket in cytochrome bd can bind one rather than two diatomic molecules. Occupancy of the site by one ligand molecule probably blocks access of a second molecule. Thus cytochrome bd exhibits strong negative cooperativity in ligand binding. Immediately after photolysis/recombination CO occupies 100% of the heme d sites, whereas after equilibration, the ligand gets located at heme d in 90–95% and at heme b595 in 5–10% of the cytochrome. The equilibration process is possibly associated with an exchange of heme d endogenous ligand.

energetic status of the amoeba cell (by decreasing the yield of ATP synthesis) and in attenuating reactive oxygen species production. AcAOX is stimulated by purine nucleotides, except ATP that has an inhibitory effect on AcAOX activity. A gene for AcAOX has been identified but there has been no direct functional evidence that it encodes cyanide-resistant mitochondrial oxidase. Using AcAOX cDNA sequence we performed relative quantification real time PCR and found that changes in AcAOX mRNA levels during growth of A. castellanii batch culture follows the pattern of changes in AcAOX protein levels and the enzyme activity. In order to confirm an enzymatic function of the AcAOX gene product, we performed oxygen consumption measurements of Escherichia coli DH5α strain transformed with pDrive containing the AcAOX gene. In transformed bacterial cells, the cyanide-resistant benzohydroxamatesensitive respiration was observed, indicating function of active AcAOX enzyme. Moreover, the activity was stimulated by GMP and inhibited by ATP, as it has been previously shown for AcAOX in isolated A. castellanii mitochondria. The AcAOX protein was also identified in transformed bacterial cells by immunodetection.

There are 3 major families of heme-copper respiratory oxygen reductases, A-, Band C-type enzymes. The eukaryotic mitochondrial cytochrome aa3 is a Type-A enzyme. A number of human pathogens contain a Type-C oxidase, also known as cytochrome cbb3 [1]. Protons, electrons and O2 must each have access to the enzyme active site, where O2 is reduced to water. We recently found the natural electron donor for cbb3 oxidase of Vibrio cholerae. The diheme cytochrome c4 can donate electrons to cytochrome cbb3, supporting steady state oxidase activity of at least 300e−1 [2]. However the sequence of electron transfers within the redox-active hemes of cytochrome cbb3 is not clear. Looking at the X-ray crystal structure of the cbb3 oxidase, subunit III (CcoP) appears to be a candidate for the initial acceptor of the electron from cytochrome c4 [3]. Subunit III contains two Nterminal transmembrane helices and a periplasmic globular domain containing two cytochrome c moieties. Subunit II (CcoO) contains one cytochrome c. Previous studies in our laboratory used point mutations to perturb each of the three cytochrome c components of the enzyme, and showed that each cytochrome c is essential for function. In this work, we examine the electron transfer pathway after performing two different procedures: 1) removing subunit III entirely, and 2) truncating just the periplasmic, hydrophilic domain. Deletion of subunit III resulted in no enzyme being found in the membrane. Deletion of just the periplasmic domain resulted in a stable complex containing subunits I and II, but oxidase activity is significantly reduced. This data shows that in V. cholera, the periplasmic domain of subunit III is required for oxidase activity as the likely entry point for electrons from cytochrome c4 and that the transmembrane helices appear to be required for stability and/or assembly. This situation is similar to that found in the A-type cytochrome c oxidases in which the hydrophilic domain of subunit II is the docking site for cytochrome c and a critical glutamic residue in subunit II is the entrance to the K-channel for protons.

The time-resolved kinetics of membrane potential generation coupled to oxidation of the fully reduced (five-electron) caa(3) cytochrome oxidase from Thermus thermophilus by oxygen was studied in a single-turnover regime. In order to calibrate the number of charges that move across the vesicle membrane in the different reaction steps, the reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) has been resolved upon photodissociation of CO from the mixed valence enzyme in the absence of oxygen. The reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) pair is resolved as a single transition with τ~40 μs. In the reaction of the fully reduced cytochrome caa(3) with oxygen, the first electrogenic phase (τ~30 μs) is linked to OO bond cleavage and generation of the P(R) state. The next electrogenic component (τ~50 μs) is associated with the P(R)→F transition and together with the previous reaction step it is coupled to translocation of about two charges across the membrane. The three subsequent electrogenic phases, with time constants of ~0.25 ms, ~1.4 ms and ~4 ms, are linked to the conversion of the binuclear center through the F→O(H)→E(H) transitions, and result in additional transfer of four charges through the membrane dielectric. This indicates that the delivery of the fifth electron from heme c to the binuclear center is coupled to pumping of an additional proton across the membrane.

SummaryInterpretation of the constantly expanding body of genomic information requires that the function of each gene be established. Here we report the genomic analysis and structural modelling of a previously uncharacterized redox‐metabolism protein UrdA (SO_4620) of Shewanella oneidensis MR‐1, which led to a discovery of the novel enzymatic activity, urocanate reductase. Further cloning and expression of urdA, as well as purification and biochemical study of the gene's product UrdA and redox titration of its prosthetic groups confirmed that the latter is indeed a flavin‐containing enzyme catalysing the unidirectional reaction of two‐electron reduction of urocanic acid to deamino‐histidine, an activity not reported earlier. UrdA exhibits both high substrate affinity and high turnover rate (Km << 10 μM, kcat = 360 s−1) and strong specificity in favour of urocanic acid. UrdA homologues are present in various bacterial genera, such as Shewanella, Fusobacterium and Clostridium, the latter including the human pathogen Clostridium tetani. The UrdA activity in S. oneidensis is induced by its substrate under anaerobic conditions and it enables anaerobic growth with urocanic acid as a sole terminal electron acceptor. The latter capability can provide the cells of UrdA‐containing bacteria with a niche where no other bacteria can compete and survive.

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is a component of respiratory electron-transport chain of various bacteria generating redox-driven transmembrane electrochemical Na(+) potential. We found that the change in Na(+) concentration in the reaction medium has no effect on the thermodynamic properties of prosthetic groups of Na(+)-NQR from Vibrio harveyi, as was revealed by the anaerobic equilibrium redox titration of the enzyme's EPR spectra. On the other hand, the change in Na(+) concentration strongly alters the EPR spectral properties of the radical pair formed by the two anionic semiquinones of FMN residues bound to the NqrB and NqrC subunits (FMN(NqrB) and FMN(NqrC)). Using data obtained by pulse X- and Q-band EPR as well as by pulse ENDOR and ELDOR spectroscopy, the interspin distance between FMN(NqrB) and FMN(NqrC) was found to be 15.3 Å in the absence and 20.4 Å in the presence of Na(+), respectively. Thus, the distance between the covalently bound FMN residues can vary by about 5 Å upon changes in Na(+) concentration. Using these results, we propose a scheme of the sodium potential generation by Na(+)-NQR based on the redox- and sodium-dependent conformational changes in the enzyme.

NADH:ubiquinone oxidoreductase (Complex I), the electron input enzyme in the respiratory chain of mitochondria and many bacteria, couples electron transport to proton translocation across the membrane. Complex I is a primary proton pump; although its proton translocation mechanism is yet to be known, it is considered radically different from any other mechanism known for redox-driven proton pumps: no redox centers have been found in its membrane domain where the proton translocation takes place. Here we studied the properties and the catalytic role of the enzyme-bound ubiquinone in the solubilized, purified Complex I from Escherichia coli. The ubiquinone content in the enzyme preparations was 1.3±0.1 per bound FMN residue. Rapid mixing of Complex I with NADH, traced optically, demonstrated that both reduction and re-oxidation kinetics of ubiquinone coincide with the respective kinetics of the majority of Fe-S clusters, indicating kinetic competence of the detected ubiquinone. Optical spectroelectrochemical redox titration of Complex I followed at 270-280nm, where the redox changes of ubiquinone contribute, did not reveal any transition within the redox potential range typical for the membrane pool, or loosely bound ubiquinone (ca. +50-+100mV vs. NHE, pH 6.8). The transition is likely to take place at much lower potentials (E(m) ≤-200mV). Such perturbed redox properties of ubiquinone indicate that it is tightly bound to the enzyme's hydrophobic core. The possibility of two ubiquinone-binding sites in Complex I is discussed.

CO photolysis from fully reduced Paracoccus denitrificans aa3-type cytochrome c oxidase in the absence of O2 was studied by time-resolved potential electrometry. Surprisingly, photo dissociation of the uncharged carbon monoxide results in generation of a small-amplitude electric potential with the same sign as the physiological charge separation during activity. The number of electrogenic events after CO photolysis depends on the state of the enzyme. CO photolysis following immediately after activation by an enzymatic turnover, showed a two-component potential development. A fast (~1.5μs) phase was followed by slower potential generation with a time constant varying from 8μs at pH 7 to 250μs at pH 10. The amplitude of the fast phase was independent of the time of incubation after enzyme activation, whereas the slower phase vanished with a time constant of ~25min. CO photolysis from enzyme that had not undergone a prior single turnover showed the fast phase, but the amplitude of the slow phase was reduced to 10–30%. The amplitude of the fast phase corresponds to charge movement of 0.83Å perpendicular to the membrane dielectric, and is independent of the time after enzyme activation. Thus it can be used as an internal ruler for normalization of the electrogenic responses of CcO. The slow phase was absent in the K354M mutant with a blocked proton-conducting K channel. We propose that CO photolysis increases the pK of the K354 residue, which results in its partial protonation, and generation of electric potential.

Escherichia coli is known to couple aerobic respiratory catabolism to ATP synthesis by virtue of the primary generators of the proton motive force—NADH dehydrogenase I, cytochrome bo3, and cytochrome bd-I. An E. coli mutant deficient in NADH dehydrogenase I, bo3 and bd-I can, nevertheless, grow aerobically on nonfermentable substrates, although its sole terminal oxidase cytochrome bd-II has been reported to be nonelectrogenic. In the current work, the ability of cytochrome bd-II to generate a proton motive force is reexamined. Absorption and fluorescence spectroscopy and oxygen pulse methods show that in the steady-state, cytochrome bd-II does generate a proton motive force with a H+/e- ratio of 0.94 ± 0.18. This proton motive force is sufficient to drive ATP synthesis and transport of nutrients. Microsecond time-resolved, single-turnover electrometry shows that the molecular mechanism of generating the proton motive force is identical to that in cytochrome bd-I. The ability to induce cytochrome bd-II biosynthesis allows E. coli to remain energetically competent under a variety of environmental conditions.

SummaryThe C‐terminus of the NuoL subunit of Complex I includes a long amphipathic α‐helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston‐like function. First, NuoL was truncated at its C‐ and N‐termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α‐helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild‐type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α‐helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α‐helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165–2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635–645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O2 by the X-ray beam. We show that the presence of O2 is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H2O+OH− and H2O) and chloride fit the experimental data.

Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme–copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.

The D-pathway in A-type cytochrome c oxidases conducts protons from a conserved aspartate on the negatively charged N-side of the membrane to a conserved glutamic acid at about the middle of the membrane dielectric. Extensive work in the past has indicated that all four protons pumped across the membrane on reduction of O2 to water are transferred via the D-pathway, and that it is also responsible for transfer of two out of the four "chemical protons" from the N-side to the binuclear oxygen reduction site to form product water. The function of the D-pathway has been discussed in terms of an apparent pKa of the glutamic acid. After reacting fully reduced enzyme with O2, the rate of formation of the F state of the binuclear heme-copper active site was found to be independent of pH up to pH~9, but to drop off at higher pH with an apparent pKa of 9.4, which was attributed to the glutamic acid. Here, we present an alternative view, according to which the pH-dependence is controlled by proton transfer into the aspartate residue at the N-side orifice of the D-pathway. We summarise experimental evidence that favours a proton pump mechanism in which the proton to be pumped is transferred from the glutamic acid to a proton-loading site prior to proton transfer for completion of oxygen reduction chemistry. The mechanism is discussed by which the proton-pumping activity is decoupled from electron transfer by structural alterations of the D-pathway. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Each of two hydrophobic subunits of Na+-translocating NADH:quinone oxidoreductase (NQR), NqrD and NqrE, contain a pair of strictly conserved cysteine residues within their transmembrane α-helices. Site-directed mutagenesis showed that substitutions of these residues in NQR of Vibrio harveyi blocked the Na+-dependent and 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive quinone reductase activity of the enzyme. However, these mutations did not affect the interaction of NQR with NADH and menadione. It was demonstrated that these conserved cysteine residues are necessary for the correct folding and/or the stability of the NQR complex. Mass and EPR spectroscopy showed that NQR from V. harveyi bears only a 2Fe-2S cluster as a metal-containing prosthetic group.