Differential effects of glutamate-286 mutations in the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo(3) ubiquinol oxidase from Escherichia coli

Both the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO(aa3)) and the closely related bo(3)-type ubiquinol oxidase from Escherichia coli (EcQO(bo3)) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO(aa3) result in long range conformational changes that influence the protein interactions with both heme a and heme a(3). Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcO(aa3) to ~0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a(3)-Cu(B) active site. In contrast, the E286C mutation of EcQO(bo3) exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcO(aa3), the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO(bo3) is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

FTIR spectroscopic characterization of the cytochrome aa3 from Acidianus ambivalens: evidence for the involvement of acidic residues in redox coupled proton translocation

The aa(3)-type quinol oxidase from Acidianus ambivalens is a divergent member of the heme-copper oxidases superfamily, namely, concerning the putative channels for intraprotein proton conduction. In this study, we used electrochemically induced FTIR difference spectroscopy to identify residues involved in redox-coupled protonation changes. In the spectral region characteristic for the nu(C=O) mode from protonated aspartic or glutamic acid side chains, a number of prominent features can be observed between 1790 and 1710 cm(-)(1), clearly indicating the reorganization or protonation of more than four protonatable residues upon electron transfer. A direct comparison of the Fourier-transform infrared difference spectra at different pH values reveals the noteworthy high pK of >8 for some of these residues, and the protonation of two of them. These acidic residues may play a role in the proton transport to the oxygen reducing site, in proton pumping pathways, or in protonation reactions concomitant with quinone reduction. Whereas the residues contributing between 1790 and 1750 cm(-)(1) have the typical position of an aspartic/glutamic acid side chain buried in the protein, a position closer to the surface is suggested for the residues contributing below approximately 1730 cm(-)(1). The possible involvement of residues contributing between 1750 and 1720 cm(-)(1) in the quinone binding site is demonstrated on the basis of experiments in the presence and absence of ubiquinone-2 and of the native electron carrier of the A. ambivalens respiratory chain, caldariella quinone. Most signals seen here are not observable in comparable spectra of typical members of the heme copper oxidase superfamily and thus reflect unique features of the enzyme from the hyperthermoacidophilic archaeon A. ambivalens.     

Kinetics of electron and proton transfer during O(2) reduction in cytochrome aa(3) from A. ambivalens: an enzyme lacking Glu(I-286)

Acidianus ambivalens is a hyperthermoacidophilic archaeon which grows optimally at approximately 80 degrees C and pH 2.5. The terminal oxidase of its respiratory system is a membrane-bound quinol oxidase (cytochrome aa(3)) which belongs to the heme-copper oxidase superfamily. One difference between this quinol oxidase and a majority of the other members of this family is that it lacks the highly-conserved glutamate (Glu(I-286), E. coli ubiquinol oxidase numbering) which has been shown to play a central role in controlling the proton transfer during reaction of reduced oxidases with oxygen. In this study we have investigated the dynamics of the reaction of the reduced A. ambivalens quinol oxidase with O(2). With the purified enzyme, two kinetic phases were observed with rate constants of 1.8&z.ccirf;10(4) s(-1) (at 1 mM O(2), pH 7.8) and 3. 7x10(3) s(-1), respectively. The first phase is attributed to binding of O(2) to heme a(3) and oxidation of both hemes forming the 'peroxy' intermediate. The second phase was associated with proton uptake from solution and it is attributed to formation of the 'oxo-ferryl' state, the final state in the absence of quinol. In the presence of bound caldariella quinol (QH(2)), heme a was re-reduced by QH(2) with a rate of 670 s(-1), followed by transfer of the fourth electron to the binuclear center with a rate of 50 s(-1). Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center. Moreover, the overall electron and proton-transfer kinetics in the A. ambivalens quinol oxidase are the same as those in the E. coli ubiquinol oxidase, which indicates that in the A. ambivalens enzyme a different pathway is used for proton transfer to the binuclear center and/or other protonatable groups in an equivalent pathway are involved. Potential candidates in that pathway are two glutamates at positions (I-80) and (I-83) in the A. ambivalens enzyme (corresponding to Met(I-116) and Val(I-119), respectively, in E. coli cytochrome bo(3)).     

Thermodynamic redox behavior of the heme centers of cbb3 heme-copper oxygen reductase from Bradyrhizobium japonicum

A comprehensive study of the thermodynamic redox behavior of the hemes from the cbb3 oxygen reductase from Bradyrhizobium japonicum was performed. This enzyme is a member of the C-type heme-copper oxygen reductase superfamily and has three subunits with six redox centers: four low-spin hemes and a high-spin heme and one copper ion, composing the site where oxygen is reduced. In this analysis, the visible spectra and redox properties of the five heme centers were deconvoluted. Their redox profiles and the pH dependence of the midpoint reduction potentials (redox-Bohr effect) were investigated. The reference reduction potentials (defined for a state where all centers are reduced) and homotropic interaction potentials were determined in the framework of a model of pairwise interacting redox centers. At pH 7.7, the reference reduction potentials for the three hemes c are 390, 300, and 220 mV, with low interaction potentials between them, weaker than -15 mV. For hemes b and b3, reference reduction potentials of 375 and 290 mV, respectively, were obtained; these two redox centers show an interaction potential weaker than -60 mV. The midpoint reduction potentials of all five hemes are pH-dependent. The study of these thermodynamic parameters is important in understanding the coupling mechanism of the redox and chemical processes during oxygen reduction. The analysis of the thermodynamic redox behavior of the cbb3 oxygen reductase contributes to the investigation of the mechanism of electron transfer and proton translocation by heme-copper oxygen reductases in general and indicates a thermodynamic coupling for the electron and proton transfer mechanisms.     

Heme-copper oxidases with modified D- and K-pathways are yet efficient proton pumps

The cytochrome aa(3)-type quinol oxidase from the archaeon Acidianus ambivalens and the ba(3)-type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme-copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H(+)/electron in a heme-copper oxidase that lacks most residues of the K- and D-channels. Also, the structure of the ba(3) oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D-pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton-pumping mechanism.     

Redox-linked conformational changes in bovine heart cytochrome c oxidase: picosecond time-resolved fluorescence studies of cyanide complex

Picosecond time-resolved fluorescence studies are carried out on cyanide-inhibited and heat-modified cytochrome c oxidase in aqueous lauryl maltoside surfactant solution, as well as in an aqueous vesicle, to understand the conformational changes associated with electron transfer and proton pumping activity of the enzyme. The tryptophan fluorescence decay profiles follow a four exponential model, which also matches the lifetime maxima obtained in a maximum entropy method analysis. The fast lifetime components are highly affected by the reduction and chemical modification of the enzyme. Changes in these lifetime components are related to the conformational changes in the vicinity of the heme centers of the enzyme. The cyanide-inhibited enzyme in the oxidized form shows a fluorescence decay profile similar to that of the native oxidized form, indicating that the conformational changes due to cyanide binding are very small. However, reduction of the cyanide-inhibited enzyme that leaves cyanide bound heme alpha3 oxidized causes a large increase in the fluorescence lifetimes, which indicates very significant conformational changes due to electron transfer to the dinuclear Cu(A) and heme alpha centers. A comparison of the tryptophan fluorescence decay of various other modified forms of the enzyme leads us to propose that the possible site of conformational coupling is located near heme alpha instead of the binuclear heme alpha3-Cu(B) center.     

Ligand Trapping by Cytochrome c Oxidase: IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER*

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe_a3³⁺–Cu_B²⁺) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe_a3³⁺–CN–Cu_B²⁺ complex. Instead the cyanide-induced transition of a preformed Fe_a3³⁺–N₃–Cu_B²⁺ to the ternary complex of Fe_a3³⁺–N₃ Cu_B²⁺–CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu_B blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu_B and that, during catalysis, ligands present at this copper center control access to the iron of heme a₃ from the bulk medium.     

Evolutionary migration of a post-translationally modified active-site residue in the proton-pumping heme-copper oxygen reductases

In the respiratory chains of aerobic organisms, oxygen reductase members of the heme-copper superfamily couple the reduction of O2 to proton pumping, generating an electrochemical gradient. There are three distinct families of heme-copper oxygen reductases: A, B, and C types. The A- and B-type oxygen reductases have an active-site tyrosine that forms a unique cross-linked histidine-tyrosine cofactor. In the C-type oxygen reductases (also called cbb3 oxidases), an analogous active-site tyrosine has recently been predicted by molecular modeling to be located within a different transmembrane helix in comparison to the A- and B-type oxygen reductases. In this work, Fourier-transform mass spectrometry is used to show that the predicted tyrosine forms a histidine-tyrosine cross-linked cofactor in the active site of the C-type oxygen reductases. This is the first known example of the evolutionary migration of a post-translationally modified active-site residue. It also verifies the presence of a unique cofactor in all three families of proton-pumping respiratory oxidases, demonstrating that these enzymes likely share a common reaction mechanism and that the histidine-tyrosine cofactor may be a required component for proton pumping.     

Effects of metal ions in the CuB center on the redox properties of heme in heme-copper oxidases: spectroelectrochemical studies of an engineered heme-copper center in myoglobin

The electrochemical properties of an engineered heme-copper center in myoglobin have been investigated by UV-visible spectroelectrochemistry. In the cyanide-bridged, spin-coupled heme-copper center in an engineered myoglobin, the presence of Zn(II) in the Cu(B) center raises the heme reduction potential from -85 to 49 mV vs NHE. However, in the cyanide-free, spin-decoupled derivative of the same protein, the presence of Zn(II) in the Cu(B) center exerts little influence on the heme reduction potentials (77 and 80 mV vs NHE, respectively, in the absence and in the presence of Zn(II)). Similar trends have also been observed when copper ion is present in the Cu(B) center, although on a smaller scale, due to reduction of Cu(II) to Cu(I) prior to heme reduction. These results show that the presence of a metal ion in the designed Cu(B) center has a significant effect on the redox potential of heme Fe only when the two metal centers are coupled through a bridging ligand between the two metal centers, indicating that spin coupling plays an important role in redox potential regulation. In addition, the presence of a single positively charged Cu(I) center in the Cu(B) center resulted in a much lower increase (16 mV) in heme reduction potential than that of two positively charged Zn(II) (118 mV). Therefore, the heme reduction potential must be lowered after the first electron transfer to reduce heme Fe(3+)-Cu(B)(2+) to Fe(3+)-Cu(B)(+). To raise the heme reduction potential to make the second electron transfer (i.e., reduction of Fe(3+)-Cu(B)(+) to Fe(2+)-Cu(B)(+)) to be favorable, most likely a proton or decoupling of the heme-copper center is needed in the heme-copper site. These findings provide a strong argument for a thermodynamic driving force basis for redox-regulated proton transfer in heme-copper oxidases.     

Redox-coupled proton transfer in the active site of cytochrome cbb3

Cytochrome cbb₃ is a distinct member of the superfamily of respiratory heme-copper oxidases, and is responsible for driving the respiratory chain in many pathogenic bacteria. Like the canonical heme-copper oxidases, cytochrome cbb₃ reduces oxygen to water and couples the released energy to pump protons across the bacterial membrane. Homology modeling and recent electron paramagnetic resonance (EPR) studies on wild type and a mutant cbb₃ enzyme [V. Rauhamäki et al. J. Biol. Chem. 284 (2009) 11301-11308] have led us to perform high-level quantum chemical calculations on the active site. These calculations bring molecular insight into the unique hydrogen bonding between the proximal histidine ligand of heme b₃ and a conserved glutamate, and indicate that the catalytic mechanism involves redox-coupled proton transfer between these residues. The calculated spin densities give insight in the difference in EPR spectra for the wild type and a recently studied E383Q-mutant cbb₃-enzyme. Furthermore, we show that the redox-coupled proton movement in the proximal cavity of cbb₃-enzymes contributes to the low redox potential of heme b₃, and suggest its potential implications for the high apparent oxygen affinity of these enzymes.     

Dynamics of the binuclear center of the quinol oxidase from Acidianus ambivalens.

We have investigated the kinetic and thermodynamic properties of carbon monoxide binding to the fully reduced quinol oxidase (cytochrome aa(3)) from the hyperthermophilic archaeon Acidianus ambivalens. After flash photolysis of CO from heme a(3), the complex recombines with an apparent rate constant of approximately 3 s(-1), which is much slower than with the bovine cytochrome c oxidase (approximately 80 s(-1)). Investigation of the CO-recombination rate as a function of the CO concentration shows that the rate saturates at high CO concentrations, which indicates that CO must bind transiently to Cu(B) before binding to heme a(3). With the A. ambivalens enzyme the rate reached 50% of its maximum level (which reflects the dissociation constant of the Cu(B)(CO) complex) at approximately 13 microM CO, which is a concentration approximately 10(3) times smaller than for the bovine enzyme (approximately 11 mM). After CO dissociation we observed a rapid absorbance relaxation with a rate constant of approximately 1.4 x 10(4) s(-1), tentatively ascribed to a heme-pocket relaxation associated with release of CO after transient binding to Cu(B). The equilibrium constant for CO transfer from Cu(B) to heme a(3) was approximately 10(4) times smaller for the A. ambivalens than for the bovine enzyme. The approximately 10(3) times smaller Cu(B)(CO) dissociation constant, in combination with the approximately 10(4) times smaller equilibrium constant for the internal CO transfer, results in an apparent dissociation constant of the heme a(3)(CO) complex which is "only" about 10 times larger for the A. ambivalens ( approximately 4 x 10(-3) mM) than for the bovine (0.3 x 10(-3) mM) enzyme. In summary, the results show that while the basic mechanism of CO binding to the binuclear center is similar in the A. ambivalens and bovine (and R. sphaeroides) enzymes, the heme-pocket dynamics of the two enzymes are dramatically different, which is discussed in terms of the different structural details of the A. ambivalens quinol oxidase and adaptation to different living conditions.     

The peroxynitrite reductase activity of cytochrome c oxidase involves a two-electron redox reaction at the heme a(3)-Cu(B) site

Fully and partially reduced forms of isolated bovine cytochrome c oxidase undergo a two-electron oxidation-reduction process with added peroxynitrite, leading to catalytic oxidation of ferrocytochrome c to ferricytochrome c. The other major reaction product is nitrite ion, 86% of the added peroxynitrite being measurably converted to this species. The reaction is inhibited in the presence of cyanide, implicating the heme a(3)-Cu(B) binuclear pair as the active site. Moreover, provided peroxynitrite is not added to excess, the reductase activity of the enzyme toward this oxidant efficiently protects other protein and detergent molecules in vitro from nitration of tyrosine residues and oxidative damage. If the enzyme is exposed to approximately 10(2)-fold excesses of peroxynitrite, then significant irreversible loss of electron transfer activity results, and the heme a(3)-Cu(B) binuclear pair no longer undergo a characteristic carbon monoxide-driven reduction. The accompanying rather small changes in the observed electronic absorption spectrum are suggestive of a modification in the vicinity of one or both hemes but probably not to the cofactors themselves.     

A tyrosine residue deprotonates during oxygen reduction by the caa3 reductase from Rhodothermus marinus

Heme-copper oxygen reductases catalyze proton translocation across the cellular membrane; this takes place during the reaction of oxygen to water. We demonstrate with attenuated total reflection-Fourier transform infrared (ATR-FTIR) difference spectroscopy that a tyrosine residue of the oxygen reductase from the thermohalophilic Rhodothermus marinus becomes deprotonated in the transition from the oxidized state to the catalytic intermediate ferryl state P(M). This tyrosine residue is most probably Y256, the helix VI tyrosine residue proposed to substitute for the D-channel glutamic acid that is absent in this enzyme. Comparison with the mitochondrial like oxygen reductase from Rhodobacter sphaeroides suggests that proton transfer from a strategically situated donor to the active site is a crucial step in the reaction mechanism of oxygen reductases.     

Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases

Oxygen reductase members of the heme-copper superfamily are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling the reduction of molecular oxygen to water to the translocation of protons across the plasma membrane. The protons required for catalysis and pumping in the oxygen reductases are derived from the cytoplasmic side of the membrane, transferred via proton-conducting channels comprised of hydrogen bond chains containing internal water molecules along with polar amino acid side chains. Recent analyses identified eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and H-families of oxygen reductases. Two proton input channels, the K-channel and the D-channel, are well established in the A-family of oxygen reductases (exemplified by the mitochondrial cytochrome c oxidases and by the respiratory oxidases from Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can be identified by the pattern of conserved polar amino acid residues within the protein. The C-family (cbb3 oxidases) is the second most abundant oxygen reductase family after the A-family, making up more than 20% of the sequences of the heme-copper superfamily. In this work, sequence analyses and structural modeling have been used to identify likely proton channels in the C-family. The pattern of conserved polar residues supports the presence of only one proton input channel, which is spatially analogous to the K-channel in the A-family. There is no pattern of conserved residues that could form a D-channel analogue or an alternative proton channel. The functional importance of the residues proposed to be part of the K-channel was tested by site-directed mutagenesis using the cbb3 oxidases from R. sphaeroides and Vibrio cholerae. Several of the residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D, and Y321F. The data strongly suggest that in the C-family only one channel functions for the delivery of both catalytic and pumped protons. In addition, it is also proposed that a pair of acidic residues, which are totally conserved among the C-family, may be part of a proton-conducting exit channel for pumped protons. The residues homologous to these acidic amino acids are highly conserved in the cNOR family of nitric oxide reductases and have previously been implicated as part of a proton-conducting channel delivering protons from the periplasmic side of the membrane to the enzyme active site in the cNOR family. It is possible that the C-family contains a homologous proton-conducting channel that delivers pumped protons in the opposite direction, from the active site to the periplasm.     

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.     

Time-resolved step-scan Fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo(3) from Escherichia coli

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B). Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.     

A cytochrome aa3-type quinol oxidase from Desulfurolobus ambivalens, the most acidophilic archaeon

Abstract Membranes of the extremely thermoacidophilic archaeon Desulfurolobus ambivalens grown under aerobic conditions contain a quinol oxidase of the cytochrome aa ₃-type as the most prominent hemoprotein. The partially purified enzyme consists of three polypeptide subunits with apparent molecular masses of 40, 27 and 20 kDa and contains two heme A molecules and one copper atom. CO difference spectra suggest one heme to be a heme a ₃-centre. The EPR spectra indicate the presence of a low-spin and a high-spin heme species. Redox titrations of the solubilized enzyme show the presence of two reduction processes, with apparent potentials of + 235 and + 330 mV. The enzyme cannot oxidize reduced cytochrome c , but rather serves as an oxidase of caldariella quinone. Due to their very simple composition, D . ambivalens cell appear as a promising candidate to study Structure-function relationships of cytochrome aa ₃ in the integral membrane state.     

Cyanide stimulated dissociation of chloride from the catalytic center of oxidized cytochrome c oxidase

A comparison of bovine cytochrome c oxidase isolated in the presence and the absence of chloride salts reveals that only enzyme isolated in the presence of chloride salts is a mixture of a complex of oxidized enzyme with chloride (CcO.Cl) and chloride-free enzyme (CcO). Using a spectrophotometric method for chloride determination, it was shown that CcO.Cl contains one chloride ion that is released into the medium by a single turnover or by cyanide binding. Chloride is bound slowly within the heme a(3)-Cu(B) binuclear center of oxidized enzyme in a manner similar to the binding of azide. The pH dependence of the dissociation constant for the formation of the CcO.Cl complex reveals that chloride binding proceeds with the uptake of one proton. With both forms of the enzyme the dependence of the rate of reaction for cyanide binding upon cyanide concentration asymptotes a limiting value indicating the existence of an intermediate. With CcO.Cl this limiting rate is 10(3) higher than the rate of the spontaneous dissociation of chloride from the binuclear center and we propose that the initial step is the coordination of cyanide to Cu(B) and in this intermediate state the rate of dissociation of chloride is substantially enhanced.     

Proton pumping mechanism of bovine heart cytochrome c oxidase

X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.