Primary structure of a novel subunit in ba3-cytochrome oxidase from Thermus thermophilus

The bax-type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3-cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).     

Proton transfer in ba3 cytochrome c oxidase from Thermus thermophilus

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 (aa₃-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 ba₃ 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 ba₃ oxidases with a focus on mechanisms of proton transfer and pumping. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba(3) cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa(3)-type oxidases. However the F intermediate in ba(3) oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P-->F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F-->O transition in ba(3) oxidase is close to that observed during the F-->O transition in the aa(3) oxidases. However, the P(R)-->F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

Proton transfer in ba(3) cytochrome c oxidase from Thermus thermophilus

The respiratory heme-copper oxidases catalyze reduction of O(2) to H(2)O, 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.     

Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from thermus thermophilus

Cytochrome c oxidase is a respiratory enzyme catalysing the energy-conserving reduction of molecular oxygen to water. The crystal structure of the ba(3)-cytochrome c oxidase from Thermus thermophilus has been determined to 2.4 A resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa. A structure-based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g. a new electron transfer pathway leading directly from Cu(A) to Cu(B). Specific features of the ba(3)-oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O(2-), OH(-) or H(2)O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c. New aspects of the proton pumping mechanism could be identified.     

High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment

The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H⁺ and e^- transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba₃-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O₂-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu_B atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe_a3 and Cu_B atoms that is best modeled as peroxide. The structure of ba₃-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba₃-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.     

Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation

We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with (15)N R (1), R (1ρ ), and {(1)H}-(15)N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54?kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo "gating" motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ( ex ) contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns-ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.     

The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 A resolution.

The structure of the CuA-containing, extracellular domain of Thermus thermophilus ba3-type cytochrome c oxidase has been determined to 1.6 A resolution using multiple X-ray wavelength anomalous dispersion (MAD). The Cu2S2 cluster forms a planar rhombus with a copper-copper distance of 2.51 +/- 0.03 A. X-ray absorption fine-structure (EXAFS) studies show that this distance is unchanged by crystallization. The CuA center is asymmetrical; one copper is tetrahedrally coordinated to two bridging cysteine thiolates, one histidine nitrogen and one methionine sulfur, while the other is trigonally coordinated by the two cysteine thiolates and a histidine nitrogen. Combined sequence-structure alignment of amino acid sequences reveals conserved interactions between cytochrome c oxidase subunits I and II.     

Simultaneous resonance Raman detection of the heme a3-Fe-CO and CuB-CO species in CO-bound ba3-cytochrome c oxidase from Thermus thermophilus. Evidence for a charge transfer CuB-CO transition

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba3-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the CuB-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba3-oxidase. We assigned the bands at 507 and 568 cm(-1) to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm(-1) showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the alpha-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa3-oxidase, are discussed in terms of the proposed coupling of the alpha-and beta-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The CuB-CO complex was identified by its nu(CO) at 2053 cm(-1) and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent CuB(I) that is located at the O2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.     

Studies on cytochrome c oxidase activity of the cytochrome c1aa3 complex from Thermus thermophilus

Cytochrome oxidase from T. thermophilus is isolated as a noncovalent complex of cytochromes c1 and aa3 in which the four redox components of aa3 appear to be associated with a single approximately 55,000-D subunit while the heme C is associated with a approximately 33,000-D peptide (Yoshida, T., Lorence, R. M., Choc, M. G., Tarr, G. E., Findling, K. L., and Fee, J. A. (1983) J. Biol. Chem. 258, 112-123). We have examined the steady state transfer of electrons from ascorbate to oxygen by cytochrome c1aa3 as mediated by horse heart, Candida krusei, and T. thermophilus (c552) cytochromes c as well as tetramethylphenylenediamine (TMPD). These mediators exhibit simple Michaelis-Menten kinetic behavior yielding Vmax and KM values characteristic of the experimental conditions. Three classes of kinetic behavior were observed and are qualitatively discussed in terms of a reaction scheme. The data show that tetramethylphenyldiamine and cytochromes c react with the enzyme at independent sites; it is suggested that cytochrome c1 may efficiently transfer electrons to cytochrome aa3. When incorporated into phospholipid vesicles, the highly purified cytochrome c1aa3 was found to translocate one proton into the exterior medium for each molecule of cytochrome c552 oxidized. The combined results suggest that this bacterial enzyme functions in a manner generally identical with the more complex eucaryotic enzyme.     

Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)⁺ with the concurrent reduction of O₂ to H₂O₂, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and ¹⁵N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.     

The Reactions of O2 and NO with Mixed-Valence ba3 Cytochrome c Oxidase from Thermus thermophilus

Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba₃ (Tt ba₃) cytochrome oxidase revealed that O₂ binding was slowed down by a factor of 10 in the presence of CO (Szundi et al., 2010, PNAS 107, 21010–21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the Cu_B⁺-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba₃ (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380–1390) affects O₂ and NO binding and the ability of Cu_B to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba₃ with O₂ were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O₂ and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba₃. The rate of O₂ and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived Cu_B⁺-CO complex did not prevent O-O bond splitting and the resulting P_M formation, which was significantly faster (5–10 times) than in the bovine heart enzyme. We propose that O₂ binding to heme a₃ in Tt ba₃ causes CO to dissociate from Cu_B⁺ in a concerted manner through steric and/or electronic effects, thus allowing Cu_B⁺ to act as an electron donor in the mixed-valence enzyme. The significantly faster O₂ binding and O-O bond cleavage in Tt ba₃ compared to analogous steps in the aa₃ oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.     

NO binding and dynamics in reduced heme-copper oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration. It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations. We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steady-state low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results. A comparative study was performed on CcO ba(3) from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity. The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa(3), whereas strong rebinding on the 15-ns time scale was observed for CcO ba(3). The EPR characterizations show similar results for aa(3) at substoichiometric NO/enzyme ratios and for ba(3), indicating formation of a 6-coordinate heme-NO complex. The presence of a second NO molecule in the aa(3) active site strongly modifies the heme-NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron. This proposal is supported by molecular-modeling studies that indicate a approximately 63 degrees rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB. It is argued that the second NO binds to CuB.     

Timing of Electron and Proton Transfer in the ba(3) Cytochrome c Oxidase from Thermus thermophilus

Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O(2) to H(2)O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba(3) oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O(2) reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H(+)/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1?H(+)/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba(3) oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a "proton-loading site" (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases.     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.     

Bioenergetics at extreme temperature: Thermus thermophilus ba3- and caa3-type cytochrome c oxidases

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 ba₃- and caa₃-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 caa₃-oxidase subunits having been initially ‘fused’. Coupled with sequence analysis, the ba₃-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.     

Potentiometric study of cytochrome c1aa3 from Thermus thermophilus

We have examined the redox behavior of the cytochrome c1aa3 complex from Thermus thermophilus. In potentiometric titrations the cytochrome c behaves as an independent center having n = 1 and E = 205 mV (NHE). Under the assumption that the individual centers equilibrate independently in this experiment, changes in the absorption band at 603 nm have been resolved into two components: cytochrome a (n = 1, Em = 270 mV, 60% spectral contribution) and cytochrome a3 (n = 2, Em = 360 mV, 40% spectral contribution). The n = 2 process was attributed to strong chemical coupling between cytochrome a3 and CuB. The enzyme was also titrated with a mixture of NADH and PMS, and the results are shown not to conform to a model of intramolecular equilibrium according to the equilibrium constants obtained from the potentiometric titration. It is suggested that a conformational equilibrium within the complex may control electron transfer between cytochromes a and a3.     

Probing the Q-proton pathway of ba3-cytochrome c oxidase by time-resolved Fourier transform infrared spectroscopy

In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases. The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool. We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature. The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)). The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed.     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

The rate-limiting step in O2 reduction by cytochrome ba3 from Thermus thermophilus

Cytochrome ba₃ (ba₃) 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 a₃) and a copper (Cu_B). 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 ba₃ 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 a₃ 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 Cu_B, prior to its migration to heme a₃ for the oxidative reaction, highlighting the critical role of Cu_B in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.