BBA - Bioenergetics (v.1817, #4)

Respiratory oxidases by Bernd Ludwig (467).

The mechanism of dioxygen activation and reduction in cell respiration, as catalysed by cytochrome c oxidase, has a long history. The work by Otto Warburg, David Keilin and Britton Chance defined the dioxygen-binding heme iron centre, viz. das Atmungsferment, or cytochrome a 3 . Chance brought the field further in the mid-1970's by ingenious low-temperature studies that for the first time identified the primary enzyme-substrate (ES) Michaelis complex of cell respiration, the dioxygen adduct of heme a 3 , which he termed Compound A. Further work using optical, resonance Raman, EPR, and other sophisticated spectroscopic techniques, some of which with microsecond time resolution, has brought us to the situation today, where major principles of how O2 reduction occurs in respiration are well understood. Nonetheless, some questions have remained open, for example concerning the precise structures, catalytic roles, and spectroscopic properties of the breakdown products of Compound A that have been called P, F (for peroxy and ferryl), and O (oxidised). This nomenclature has been known to be inadequate for some time already, and an alternative will be suggested here. In addition, the multiple forms of P, F and O states have been confusing, a situation that we endeavour to help clarifying. The P and F states formed artificially by reacting cytochrome oxidase with hydrogen peroxide are especially scrutinised, and some novel interpretations will be given that may account for previously unexplained observations. This article is part of a Special Issue entitled: Respiratory Oxidases.
Keywords: Cell respiration; Oxygen reduction; Heme–copper oxidase;

The paper presents a survey of time-resolved studies of charge translocation by cytochrome c oxidase coupled to transfer of the 1st, 2nd 3rd and 4th electrons in the catalytic cycle. Single-electron photoreduction experiments carried out with the A-class cytochrome c oxidases of aa 3 type from mitochondria, Rhodobacter sphaeroides and Paracoccus denitrificans as well as with the ba 3-type oxidase from Thermus thermophilus indicate that the protonmotive mechanisms, although similar, may not be identical for different partial steps in the same enzyme species, as well as for the same single-electron transition in different oxidases. The pattern of charge translocation coupled to transfer of a single electron in the A-class oxidases confirms major predictions of the original model of proton pumping by cytochrome oxidase [Artzatbanov, V. Y., Konstantinov, A. A. and Skulachev, V.P. “Involvement of Intramitochondrial Protons in Redox Reactions of Cytochrome a.” FEBS Lett. 87: 180–185]. The intermediates and partial electrogenic steps observed in the single-electron photoreduction experiments may be very different from those observed during oxidation of the fully reduced oxidase by O2 in the “flow-flash” studies. This article is part of a Special Issue entitled: Respiratory Oxidases.► Charge transfer coupled to single-electron transitions of COX is reviewed. ► Pattern of charge transfer by COX differs for the 4 single e transitions. ► Electrogenic mechanisms are similar for the 3rd and 4th e in bovine. COX. ► Electrogenic mechanisms are different for the 3rd and 4th e in bacterial COX. ► ET from CuA to heme a may be coupled to internal H+ transfer steps.
Keywords: Cytochrome oxidase; Proton pumping; Time-resolved electrometric study; Energy transduction; Cytochrome aa3; Cytochrome ba3;

Gating and regulation of the cytochrome c oxidase proton pump by Shelagh Ferguson-Miller; Carrie Hiser; Jian Liu (489-494).
As a consumer of 95% of the oxygen we breathe, cytochrome c oxidase plays a major role in the energy balance of the cell. Regulation of its oxygen reduction and proton pumping activity is therefore critical to physiological function in health and disease. The location and structure of pathways for protons that are required to support cytochrome c oxidase activity are still under debate, with respect to their requirements for key residues and fixed waters, and how they are gated to prevent (or allow) proton backflow. Recent high resolution structures of bacterial and mammalian forms reveal conserved lipid and steroid binding sites as well as redox-linked conformational changes that provide new insights into potential regulatory ligands and gating modes. Mechanistic interpretation of these findings and their significance for understanding energy regulation is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases.► Cytochrome c oxidase is a critical player in regulation of aerobic metabolism. ► Lipid binding sites are conserved, indicating structural and functional roles. ► Conformational change is involved in regulation of proton uptake in K path. ► Frozen crystals maintain structure even when reduced by X-rays.
Keywords: Cytochrome aa3; Conformational change; Bile salts; Lipid binding;

The mechanism for proton pumping in cytochrome c oxidase in the respiratory chain, has for decades been one of the main unsolved problems in biochemistry. However, even though several different suggested mechanisms exist, many of the steps in these mechanisms are quite similar and constitute a general consensus framework for discussing proton pumping. When these steps are analyzed, at least three critical gating situations are found, and these points are where the suggested mechanisms in general differ. The requirements for gating are reviewed and analyzed in detail, and a mechanism is suggested, where solutions for all the gating situations are formulated. This mechanism is based on an electrostatic analysis of a kinetic experiment fior the O to E transition. The key component of the mechanism is a positively charged transition state. An electron on heme a opens the gate for proton transfer from the N-side to a pump loading site (PLS). When the negative charge of the electron is compensated by a chemical proton, the positive transition state prevents backflow from the PLS to the N-side at the most critical stage of the pumping process. The mechanism has now been tested by large model DFT calculations, and these calculations give strong support for the suggested mechanism. This article is part of a Special Issue entitled: Respiratory Oxidases.► We review different suggestions for proton pumping mechanisms. ► We suggest one mechanism taking care of all important gating situations. ► We report quantum chemical calculations on large models supporting the suggested mechanism.
Keywords: Cytochrome c oxidase; Proton pumping; Gating; Quantum chemistry; DFT;

A combined DFT/electrostatic approach is employed to study the coupling of proton and electron transfer reactions in cytochrome c oxidase (CcO) and its proton pumping mechanism. The coupling of the chemical proton to the internal electron transfer within the binuclear center is examined for the O → E transition. The novel features of the His291 pumping model are proposed, which involve timely well-synchronized sequence of the proton-coupled electron transfer reactions. The obtained pK a s and E m s of the key ionizable and redox-active groups at the different stages of the O → E transition are consistent with available experimental data. The PT step from E242 to H291 is examined in detail for various redox states of the hemes and various conformations of E242 side-chain. Redox potential calculations of the successive steps in the reaction cycle during the O → E transition are able to explain a cascade of equilibria between the different intermediate states and electron redistribution between the metal centers during the course of the catalytic activity. All four electrometric phases are discussed in the light of the obtained results, providing a robust support for the His291 model of proton pumping in CcO. This article is part of a Special Issue entitled: Respiratory oxidases.Display Omitted► DFT/electrostatic method is used to study proton pumping in cytochrome oxidase. ► Novel features of His291 pumping model are considered. ► The coupling of PT from Glu242 to His291 to ET between the hemes is discussed. ► The pK a s and E m s of the key ionizable and redox groups are evaluated.
Keywords: Cytochrome c oxidase; Donor–acceptor system; Gating mechanism; pK a and E m calculation; Proton-loading site; Proton pumping;

In cytochrome c oxidase (CcO), a redox-driven proton pump, protons are transported by the Grotthuss shuttling via hydrogen-bonded water molecules and protonatable residues. Proton transport through the D-pathway is a complicated process that is highly sensitive to alterations in the amino acids or the solvation structure in the channel, both of which can inhibit proton pumping and enzymatic activity. Simulations of proton transport in the hydrophobic cavity showed a clear redox state dependence. To study the mechanism of proton pumping in CcO, multi-state empirical valence bond (MS-EVB) simulations have been conducted, focusing on the proton transport through the D-pathway and the hydrophobic cavity next to the binuclear center. The hydration structures, transport pathways, effects of residues, and free energy surfaces of proton transport were revealed in these MS-EVB simulations. The mechanistic insight gained from them is herein reviewed and placed in context for future studies. This article is part of a Special Issue entitled: Respiratory Oxidases.► Reactive molecular dynamics studies of proton transport in CcO are reported. ► The multi-state empirical valence bond (MS-EVB) methodology is used. ► Proton shuttling mechanism through water molecules in CcO was studied. ► Proton transport in the D-channel and the hydrophobic cavity past E286 were studied. ► We provide molecular insight how the electronic state modulates the proton transport.
Keywords: Cytochrome c oxidase; Proton pump; Proton transport; Computer simulation; Multi-state empirical valence bond (MS-EVB);

Cytochrome c oxidase is an efficient energy transducer that reduces oxygen to water and converts the released chemical energy into an electrochemical membrane potential. As a true proton pump, cytochrome c oxidase translocates protons across the membrane against this potential. Based on a wealth of experiments and calculations, an increasingly detailed picture of the reaction intermediates in the redox cycle has emerged. However, the fundamental mechanism of proton pumping coupled to redox chemistry remains largely unresolved. Here we examine and extend a kinetic master-equation approach to gain insight into redox-coupled proton pumping in cytochrome c oxidase. Basic principles of the cytochrome c oxidase proton pump emerge from an analysis of the simplest kinetic models that retain essential elements of the experimentally determined structure, energetics, and kinetics, and that satisfy fundamental physical principles. The master-equation models allow us to address the question of how pumping can be achieved in a system in which all reaction steps are reversible. Whereas proton pumping does not require the direct modulation of microscopic reaction barriers, such kinetic gating greatly increases the pumping efficiency. Further efficiency gains can be achieved by partially decoupling the proton uptake pathway from the active-site region. Such a mechanism is consistent with the proposed Glu valve, in which the side chain of a key glutamic acid shuttles between the D channel and the active-site region. We also show that the models predict only small proton leaks even in the absence of turnover. The design principles identified here for cytochrome c oxidase provide a blueprint for novel biology-inspired fuel cells, and the master-equation formulation should prove useful also for other molecular machines. This article is part of a Special Issue entitled: Respiratory Oxidases.► Kinetic-master equations model proton pumping in cytochrome c oxidase. ► Proton pumping at low efficiency can be achieved without kinetic gating. ► High thermodynamic efficiency of pumping requires kinetic gating. ► Internal proton transfer to pump site is key gated reaction step. ► Glu valve at the end of the D channel increases the pumping efficiency.
Keywords: Proton pumping; Cytochrome c oxidase; Respiratory chain; Energy transduction; Molecular machine; Kinetic master equation;

Functional proton transfer pathways in the heme–copper oxidase superfamily by Hyun Ju Lee; Joachim Reimann; Yafei Huang; Pia Ädelroth (537-544).
Heme–copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review. This article is part of a Special Issue entitled: Respiratory Oxidases.► We describe proton transfer pathways in A, B, C oxidases as well as cNORs. ► B and C oxidases use only one proton pathway. ► In C-type, this pathway starts at a Glu in CcoP. ► We briefly review proton pumping mechanisms across the superfamily.
Keywords: cbb 3; aa 3; ba 3; NOR; K-pathway; Oxygen reduction;

We review studies of subunit III-depleted cytochrome c oxidase (CcO III (−)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O2 consumption can be used to compare the rate and functional pK a of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (−) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake. This article is part of a Special Issue entitled: Respiratory Oxidases.► Subunit III-depleted cytochrome c oxidase provides information about proton uptake. ► Steady-state activity compares proton uptake via different initial proton acceptors. ► Catalytic lifespan is another method to compare different proton uptake strategies.
Keywords: Cytochrome c oxidase; Proton uptake; Proton transfer; D pathway; Proton acceptor;

The origin of the FeIV  = O intermediates in cytochrome aa 3 oxidase by Eftychia Pinakoulaki; Vangelis Daskalakis; Constantinos Varotsis (552-557).
The dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P → F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a 3–propionateA–D399–H403 chain. This article is part of a Special Issue entitled: Respiratory Oxidases.Display Omitted► The origin of the ferryl intermediates is addressed. ► Proton transfer mechanisms are discussed. ► Proton translocation (P → F) is related to two groups of conserved residues.
Keywords: Heme–copper oxidase; Ferryl intermediate; Resonance Raman spectroscopy; Molecular dynamics simulation;

Allosteric interactions and proton conducting pathways in proton pumping aa 3 oxidases: Heme a as a key coupling element by Nazzareno Capitanio; Luigi Leonardo Palese; Giuseppe Capitanio; Pietro Luca Martino; Oliver-Matthias H. Richter; Bernd Ludwig; Sergio Papa (558-566).
In this paper allosteric interactions in protonmotive heme aa 3 terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H+/e coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa 3 oxidase, which decreases by more than 200 mV the Em of heme a, inhibits proton pumping. Mutational aminoacid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa 3 oxidases, as well as Zn2 + binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O2 to 2 H2O. This article is part of a Special Issue entitled: Respiratory Oxidases.► Characterization of allosteric interactions in proton pumping aa 3 oxidases. ► Heme a plays a key role in the proton pump of aa 3 oxidases. ► Aminoacid replacement or Zn2 + binding at the negative surface of COV uncouple proton pumping.
Keywords: aa 3 terminal oxidase; Redox proton pumping; Redox Bohr effect;

This review describes the development and application of photoactive ruthenium complexes to study electron transfer and proton pumping reactions in cytochrome c oxidase (CcO). CcO uses four electrons from Cc to reduce O2 to two waters, and pumps four protons across the membrane. The electron transfer reactions in cytochrome oxidase are very rapid, and cannot be resolved by stopped-flow mixing techniques. Methods have been developed to covalently attach a photoactive tris(bipyridine)ruthenium group [Ru(II)] to Cc to form Ru-39-Cc. Photoexcitation of Ru(II) to the excited state Ru(II*), a strong reductant, leads to rapid electron transfer to the ferric heme group in Cc, followed by electron transfer to CuA in CcO with a rate constant of 60,000 s− 1. Ruthenium kinetics and mutagenesis studies have been used to define the domain for the interaction between Cc and CcO. New ruthenium dimers have also been developed to rapidly inject electrons into CuA of CcO with yields as high as 60%, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism. This article is part of a Special Issue entitled: Respiratory Oxidases.► A variety of ruthenium based flash photolysis schemes are described. ► Rate of electron transfer between cytochrome c and cytochrome oxidase is measured. ► Binding domain between cytochrome c and cytochrome c oxidase is defined. ► Rate of electron transfer between CuA and heme a is measured. ► Rate of electron transfer between heme a and heme a3/CuB is measured.
Keywords: Cytochrome c oxidase; Ruthenium; Kinetics; Electron transfer; Proton pumping;

Recent applications of resonance Raman (RR) spectroscopy in investigations of cytochrome c oxidase (CcO) are reviewed. Red-excited RR spectra for the fully oxidized “as-isolated” CcO tuned to the ligand-to-metal charge transfer absorption band at 655 nm exhibit a Raman band at 755 cm− 1 assignable to the ν OO stretching mode of a peroxide. Binding of CN diminishes the RR band concomitant with the loss of the charge transfer absorption band. This suggests that a peroxide forms a bridge between heme a 3 and CuB. Time-resolved RR spectroscopy of whole mitochondria identified a band at 571 cm− 1 arising from the oxygenated intermediate at Δt  = 0.4, 0.6 and 1.4 ms. Bands at 804 and 780 cm− 1 of the P and F intermediates were observed at Δt  = 0.6 and 1.4 ms, respectively. The coordination geometries of the three intermediates are essentially the same as the respective species observed for solubilized CcO. However, the lifetime of the oxygenated intermediate in mitochondria was significantly longer than the lifetime of this intermediate determined for solubilized CcO. This phenomenon is due either to the pH effect of mitochondrial matrix, the effect of ΔpH and/or ΔΨ across the membrane, or the effect of interactions with other membrane components and/or phospholipids. This article is part of a Special Issue entitled: Respiratory Oxidases.► Resonance Raman (RR) spectroscopy was applied to cytochrome c oxidase (CcO). ► The fully-oxidized “as-isolated” CcO was revealed to have the Fe―O2 2 −―Cu center. ► Oxygen activation by CcO in situ in mitochondria could be studied by RR spectroscopy. ► CcO in situ and solubilized CcO shared essentially the same reaction intermediates. ► The oxygenated intermediate in situ decayed much slower than for solubilized CcO.
Keywords: Cytochrome c oxidase; Proton pump; Oxygen activation; Resonance Raman; Mitochondria;

Structural studies on bovine heart cytochrome c oxidase by Shinya Yoshikawa; Kazumasa Muramoto; Kyoko Shinzawa-Itoh; Masao Mochizuki (579-589).
Among the X-ray structures of bovine heart cytochrome c oxidase (CcO), reported thus far, the highest resolution is 1.8 Å. CcO includes 13 different protein subunits, 7 species of phospholipids, 7 species of triglycerides, 4 redox-active metal sites (CuA, heme a (Fe a ), CuB, heme a 3 (Fe a3)) and 3 redox-inactive metal sites (Mg2 +, Zn2 + and Na+).The effects of various O2 analogs on the X-ray structure suggest that O2 molecules are transiently trapped at the CuB site before binding to Fe a3 2 + to provide O2 . This provides three possible electron transfer pathways from CuB, Fe a3 and Tyr244 via a water molecule. These pathways facilitate non-sequential 3 electron reduction of the bound O2 to break the O―O bond without releasing active oxygen species.Bovine heart CcO has a proton conducting pathway that includes a hydrogen-bond network and a water-channel which, in tandem, connect the positive side phase with the negative side phase. The hydrogen-bond network forms two additional hydrogen-bonds with the formyl and propionate groups of heme a. Thus, upon oxidation of heme a, the positive charge created on Fe a is readily delocalized to the heme peripheral groups to drive proton-transport through the hydrogen-bond network. A peptide bond in the hydrogen-bond network and a redox-coupled conformational change in the water channel are expected to effectively block reverse proton transfer through the H-pathway. These functions of the pathway have been confirmed by site-directed mutagenesis of bovine CcO expressed in HeLa cells. This article is part of a Special Issue entitled: Respiratory Oxidases.► Metal, lipid and protein assembly of bovine cytochrome c oxidase. ► O2 reduction by O2 binding to Fe2 +, followed by non-sequential 3-electron reduction. ► Positive charges of low spin heme, upon oxidation, pump protons electrostatically. ► X-ray and mutational identification of proton pump system of cytochrome c oxidase.
Keywords: X-ray structural analyses; Membrane proteins; Hemoproteins; Bovine cytochrome c oxidase; O2 reduction; Proton pump;

Cytochrome c oxidase: Evolution of control via nuclear subunit addition by Denis Pierron; Derek E. Wildman; Maik Hüttemann; Gopi Chand Markondapatnaikuni; Siddhesh Aras; Lawrence I. Grossman (590-597).
According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the “higher” organism possessing large brains and muscles. The main function of these subunits appears to be “only” to control the activity of the mitochondrial subunits. We propose that this control function is an as yet underappreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a “domestication scenario” such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. This article is part of a Special Issue entitled: Respiratory Oxidases.► OXPHOS allowscells to produce more ATP but also toxic elements such as ROS and NADH. ► Success of endosymbiosis has depended on the regulation of endosymbiont OXPHOS. ► Cytochrome c oxidase (COX) is a key regulator of OXPHOS. ► Addition of nuclear subunits controlling mitochondrial subunits marks COX evolution. ► COX control function is an as yet under-appreciated key point of evolution.
Keywords: Evolution; Oxidative phosphorylation; Mitochondria; Endosymbiosis; Regulation;

Regulation of mitochondrial respiration and apoptosis through cell signaling: Cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation by Maik Hüttemann; Stefan Helling; Thomas H. Sanderson; Christopher Sinkler; Lobelia Samavati; Gargi Mahapatra; Ashwathy Varughese; Guorong Lu; Jenney Liu; Rabia Ramzan; Sebastian Vogt; Lawrence I. Grossman; Jeffrey W. Doan; Katrin Marcus; Icksoo Lee (598-609).
Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis. This article is part of a Special Issue entitled: “Respiratory Oxidases”.Display Omitted► Cytochrome c oxidase (COX) activity is controlled by cell signaling pathways. ► Cell signaling leads to changes of the phosphorylation state of COX. ► COX activity controls the mitochondrial membrane potential, energy, and ROS production. ► During acute inflammation COX is hypoactive leading to an energy crisis and cell death. ► Ischemia/reperfusion causes COX hyperactivation, excessive ROS, and apoptosis.
Keywords: Apoptosis; Cancer; Neurodegenerative disease; Oxidative phosphorylation; Sepsis; Stroke;

Cytochrome c oxidase and nitric oxide in action: Molecular mechanisms and pathophysiological implications by Paolo Sarti; Elena Forte; Daniela Mastronicola; Alessandro Giuffrè; Marzia Arese (610-619).
The reactions between Complex IV (cytochrome c oxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondrial Complex I and IV particularly, found responsible of major effects.The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants.The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO2 ) of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O2. All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells. This article is part of a Special Issue entitled: Respiratory Oxidases.► Nitric oxide reacts with the metals of the binuclear site of cytochrome c oxidase which is inhibited. ► A nitrosyl-derivative (CcOX-NO) or a nitrite-derivative (CcOX-NO2 ) can be formed. ► Persistence of inhibition depends on the prevailing mechanism of reaction. ► Substrates availability, O2 and cytochrome c 2+, and the NO concentration control the mechanism. ► Inhibition is O2-competitive only if CcOX nitrosylation occurs.
Keywords: Respiratory chain; Radical chemistry; Mitochondria; Electron transfer; Hemeproteins; Enzyme inhibition;

Yeast cytochrome c oxidase: A model system to study mitochondrial forms of the haem–copper oxidase superfamily by Amandine Maréchal; Brigitte Meunier; David Lee; Christine Orengo; Peter R. Rich (620-628).
The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed. This article is part of a Special Issue entitled: Respiratory Oxidases.► The structure of the 11 subunit yeast cytochrome c oxidase was homology modelled. ► Three possible proton pathways were evident in the modelled structure of Cox1. ► Differences in the H channel are discussed. ► Methods to produce mutations in mtDNA-encoded core subunits are reviewed.
Keywords: Mitochondria; Cytochrome c oxidase; Complex IV; Yeast; Mutants; Supernumerary subunits;

The superfamily of heme–copper oxygen reductases: Types and evolutionary considerations by Filipa L. Sousa; Renato J. Alves; Miguel A. Ribeiro; José B. Pereira-Leal; Miguel Teixeira; Manuela M. Pereira (629-637).
Heme–copper oxygen reductases (HCO) reduce O2 to water being the last enzymatic complexes of most aerobic respiratory chains. These enzymes promote energy conservation coupling the catalytic reaction to charge separation and charge translocation across the prokaryotic cytoplasmatic or mitochondrial membrane. In this way they contribute to the establishment and maintenance of the transmembrane difference of electrochemical potential, which is vital for solute/nutrient cell import, synthesis of ATP and motility. The HCO enzymes most probably share with the nitric oxide reductases, NORs, a common ancestor. We have proposed the classification of HCOs into three different types, A, B and C; based on the constituents of their proton channels (Pereira, Santana and Teixeira (2001) Biochim Biophys Acta, 1505, 185–208). This classification was recently challenged by the suggestion of other different types of HCOs. Using an enlarged sampling we performed an exhaustive bioinformatic reanalysis of HCOs family. Our results strengthened our previously proposed classification and showed no need for the existence of more divisions. Now, we analyze the taxonomic distribution of HCOs and NORs and the congruence of their sequence trees with the 16S rRNA tree. We observed that HCOs are widely distributed in the two prokaryotic domains and that the different types of enzymes are not confined to a specific taxonomic group or environmental niche. This article is part of a Special Issue entitled: Respiratory Oxidases.► We have proposed the classification of HCOs into three different types, A, B and C. ► The classification allows probing the relevance of the proposed catalytic mechanisms. ► HCOs are widely distributed in the two prokaryotic domains. ► Different HCOs are not confined to specific taxonomic groups or environmental niches.
Keywords: Respiration; Electron transfer chain; Oxygen; Cytochrome c oxidase; Quinol oxidase;

Seven years into the completion of the genome sequencing projects of the thermophilic bacterium Thermus thermophilus strains HB8 and HB27, many questions remain on its bioenergetic mechanisms. A key fact that is occasionally overlooked is that oxygen has a very limited solubility in water at high temperatures. The HB8 strain is a facultative anaerobe whereas its relative HB27 is strictly aerobic. This has been attributed to the absence of nitrate respiration genes from the HB27 genome that are carried on a mobilizable but highly-unstable plasmid. In T. thermophilus, the nitrate respiration complements the primary aerobic respiration. It is widely known that many organisms encode multiple biochemically-redundant components of the respiratory complexes. In this minireview, the presence of the two cytochrome c oxidases (CcO) in T. thermophilus, the ba 3- and caa 3-types, is outlined along with functional considerations. We argue for the distinct evolutionary histories of these two CcO including their respective genetic and molecular organizations, with the caa 3-oxidase subunits having been initially ‘fused’. Coupled with sequence analysis, the ba 3-oxidase crystal structure has provided evolutionary and functional information; for example, its subunit I is more closely related to archaeal sequences than bacterial and the substrate–enzyme interaction is hydrophobic as the elevated growth temperature weakens the electrostatic interactions common in mesophiles. Discussion on the role of cofactors in intra- and intermolecular electron transfer and proton pumping mechanism is also included. This article is part of a Special Issue entitled: Respiratory Oxidases.► T. thermophilus ba 3- and caa 3-type cytochrome c oxidases have evolved separately. ► The subunits of caa 3-oxidase and its heme insertion factor are fused. ► The fused genes are phylogenetically ancient. ► The subunit IV of caa 3- and aa 3-oxidases is not evolutionarily conserved. ► The ba 3-oxidase has certain structural features dissimilar to aa 3-oxidases.
Keywords: ba 3-oxidase; caa 3-oxidase; Respiratory complex; Thermus thermophilus;

Proton transfer in ba 3 cytochrome c oxidase from Thermus thermophilus by Christoph von Ballmoos; Pia Ädelroth; Robert B. Gennis; Peter Brzezinski (650-657).
The respiratory heme-copper oxidases catalyze reduction of O2 to H2O, linking this process to transmembrane proton pumping. These oxidases have been classified according to the architecture, location and number of proton pathways. Most structural and functional studies to date have been performed on the A-class oxidases, which includes those that are found in the inner mitochondrial membrane and bacteria such as Rhodobacter sphaeroides and Paracoccus denitrificans (aa 3-type oxidases in these bacteria). These oxidases pump protons with a stoichiometry of one proton per electron transferred to the catalytic site. The bacterial A-class oxidases use two proton pathways (denoted by letters D and K, respectively), for the transfer of protons to the catalytic site, and protons that are pumped across the membrane. The B-type oxidases such as, for example, the ba 3 oxidase from Thermus thermophilus, pump protons with a lower stoichiometry of 0.5 H+/electron and use only one proton pathway for the transfer of all protons. This pathway overlaps in space with the K pathway in the A class oxidases without showing any sequence homology though. Here, we review the functional properties of the A- and the B-class ba 3 oxidases with a focus on mechanisms of proton transfer and pumping. This article is part of a Special Issue entitled: Respiratory Oxidases.Display Omitted► Review on recent findings in the ba 3 cytochrome c oxidase from T. thermophilus. ► Explains the 50% (average 0.5 H+/electron) pumping stoichiometry in these enzymes. ► The uptake and release of pumped protons is resolved in time.
Keywords: Respiratory oxidase; Electron transfer; Energy conservation; Electrochemical gradient; Kinetics; Membrane protein;

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a 3 moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba 3-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fea3–CO and appearance of CuB–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba 3–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm− 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm− 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm− 1, 1977 cm− 1, 1981 cm− 1, and 1985 cm− 1, previously assigned to the Fea3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm− 1, previously assigned to the CO stretching frequency of CuB bound CO. As expected for light propagating along the tetragonal axis of the P43212 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba 3 crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO2 at 2337 cm− 1 and one from traces of CO at 2133 cm− 1; while bands associated with CO bound to either Fea3 or to CuB in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fea3–CO in cytochrome c oxidases leads to significant trapping of the CO on the CuB atom; Fea3 and CuB ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.
Keywords: Cytochrome c oxidase; Cytochrome ba 3 oxidase; Carbon monoxide; CO photodissociation; Thermus thermophilus; Fourier transform infrared;

The rate-limiting step in O2 reduction by cytochrome ba 3 from Thermus thermophilus by Tsuyoshi Egawa; Ying Chen; James A. Fee; Syun-Ru Yeh; Denis L. Rousseau (666-671).
Cytochrome ba 3 (ba 3) of Thermus thermophilus (T. thermophilus) is a member of the heme–copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a 3) and a copper (CuB). The heme–copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba 3 with O2 using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~ 1 ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k  = 400 s− 1) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1 ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O2 binds rapidly to heme a 3 in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O2 binds transiently to a trap, likely CuB, prior to its migration to heme a 3 for the oxidative reaction, highlighting the critical role of CuB in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.Display Omitted► Cytochrome ba 3 (ba 3) of T. thermophilus is a member of the heme–copper oxidase family. ► ba 3 has a binuclear catalytic center comprised of a heme (heme a 3) and a copper (CuB). ► The kinetics of the reaction of the enzyme with oxygen has a fast and a slow phase. ► The slow phase of the reaction is attributed to transient binding of oxygen to CuB.
Keywords: Cytochrome oxidase; Bioenergetics; Raman scattering; Stopped-flow;

Kinetic studies of the reactions of O2 and NO with reduced Thermus thermophilus ba 3 and bovine aa 3 using photolabile carriers by Ólöf Einarsdóttir; Chie Funatogawa; Tewfik Soulimane; Istvan Szundi (672-679).
The reactions of molecular oxygen (O2) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba 3 and bovine heart aa 3 were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O2 and NO complexes. The binding of O2/NO to reduced ba 3 in the absence of CO occurs with a second-order rate constant of 1 × 109  M− 1  s− 1. This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O2/NO binding in ba 3 is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O2 and NO access to the active site in Tt ba 3, and that traditional CO flow-flash experiments do not accurately reflect the O2 and NO binding kinetics in ba 3. We suggest that in ba3 the binding of O2 (NO) to heme a 3 2 + causes rapid dissociation of CO from CuB + through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to CuB +. These findings indicate that structural differences between Tt ba 3 and the bovine aa 3 enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.
Keywords: Thermus thermophilus ba 3; O2 and NO photolabile carriers; Double-laser transient absorption spectroscopy; CO photodissociation;

Molecular structure and function of bacterial nitric oxide reductase by Tomoya Hino; Shingo Nagano; Hiroshi Sugimoto; Takehiko Tosha; Yoshitsugu Shiro (680-687).
The crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b 3 and non-heme FeB). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure. Glu211 and Glu280 appear to play an important role in the catalytic reduction of NO at the binuclear center by functioning as a terminal proton donor, while Glu215 probably contributes to the electro-negative environment of the catalytic center. Glu135, a ligand for Ca2+ sandwiched between two heme propionates from heme b and b 3, and the nearby Glu138 appears to function as a structural factor in maintaining a protein conformation that is suitable for electron-coupled proton transfer from the periplasmic region to the active site. On the basis of these observations, the possible molecular mechanism for the reduction of NO by cNOR is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases.► Possible roles of five well-conserved glutamic acid resides in bacterial nitric oxide reductase is discussed on the basis of its crystal structure. ► Possible mechanism of the NO reduction by this anaerobic respiratory enzyme is also proposed. ► These will provide us insight into molecular evolution of the respiratory enzymes.
Keywords: Nitric oxide reductase; Respiratory enzyme; Denitrification; Nitrous oxide;