Distance between the visible copper and cytochrome a in bovine heart cytochrome oxidase

Electron paramagnetic resonance (EPR) at 15 K was used to probe the magnetic interaction between the visible copper CuA2+ and ferric cytochrome a in the carbon monoxide compound of beef heart cytochrome oxidase. At pH 8.6, the midpoint potentials (Em's) for one-electron oxidation of CuA+ and cytochrome a2+ were found to be 195 and 235 mV, respectively. Because the Em of CuA is well below that of cytochrome a under these conditions, the microwave power saturation of CuA could be measured as a function of percentage cytochrome a oxidized. Although progressive power saturation data directly provide only the product of the spin-lattice and transverse relaxation rates delta [1/(T1T2)], Castner's theory for the saturation of inhomogeneously broadened lines [Castner, T.G., Jr. (1959) Phys. Rev. 115 (6), 1506-1515], along with our own theoretical formulation of the dipolar T2, enabled us to determine the change in T1 of CuA due to dipolar relaxation by cytochrome a. The orientation of the principal g values of CuA with respect to those of cytochrome a was evaluated in partially oriented membranous multilayers. When allowance was made for uncertainties in the relative CuA-cytochrome a configuration and in the dipolar axis-magnetic field orientation, a range for the spin-spin distance r was calculated on the basis of the dipolar T1 of the gx component of CuA. This distance range was further restricted by consideration of T1 for the nonunique orientations of CuA giving rise to the gy signal. Only those values of r are possible for which the calculated T1 ratio (gx/gy) is equal to the experimentally determined ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron spin-lattice relaxation of the [Cu(1.5) ... Cu(1.5)] dinuclear copper center in nitrous oxide reductase.

Relaxation times have been obtained with time-domain EPR for the dinuclear mixed valence [CuA(1.5) ... CuA(1.5)[ S = 1/2 center in nitrous oxide reductase, N2OR, from Pseudomonas stutzeri, in the TN5 mutant defective in copper chromophore biosynthesis, in a synthetic mixed valence complex, and in type 1 and 2 copper complexes. Data confirmed that the intrinsic electron spin-lattice relaxation time, T1, for N2OR in the temperature range of 6-25 K is unusually short for copper centers. At best, a twofold increase of T1 from g perpendicular to g parallel was measured. Optimized fits of the saturation-recovery data were obtained using both double-exponential and stretched-exponential functions. The temperature dependence of the spin-lattice relaxation rate of mutant N2OR is about T5.0 with the stretched-exponential model or T3.3 and T3.9 for the model using the sum of two exponentials. These T1s are intrinsic to the mixed valence [CuA(1.5) ... CuA(1.5)] center, and no interaction of the second copper center in wild-type N2OR with the [CuA(1.5) ... CuA(1.5)] center has been observed. The T1 of the mixed valence center of N2OR is not only shorter than for monomeric square planar Cu(II) complexes, but also shorter than for a synthetic mixed valence complex, Cu2(N[CH2CH2NHCH2CH2NHCH2CH2]3N). The short T1 is attributed to the vibrational modes of type 1 copper and/or the metal-metal interaction in [CuA(1.5) ... CuA(1.5)].     

Heat treatment of cytochrome c oxidase perturbs the CuA site and affects proton pumping behavior

It has been previously reported that mild heat treatment (43 degrees C for ca. 60 min) abolishes the proton pumping activity of cytochrome c oxidase while leaving the oxidase activity and cytochromes a and a3 unperturbed [Sone, N., & Nicholls, P. (1984) Biochemistry 23, 6550-6554]. We herein describe the effects of this heat treatment on the electron paramagnetic resonance (EPR) and optical absorption signatures of the redox-active metal centers in the enzyme. We find that heat treatment of the oxidized enzyme causes a local structural perturbation at the CuA site. After heat treatment, the enzyme sample contains three subpopulations, each of which has a different structure at CuA. These include (i) native CuA, (ii) a type 2 copper species similar to the one produced by chemical modification by p-(hydroxymercuri)benzoate (pHMB) [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972], and (iii) a novel type 1 copper species. In addition to changes at the CuA site, we find that heat treatment results in accelerated cyanide binding and the removal of subunit III. If the cytochrome c oxidase is heat treated while fully reduced, none of these changes are observed except for subunit III depletion. Furthermore, partial (CO mixed-valence derivative) reduction of the enzyme as well as ligand binding to cytochrome a3 also protects the enzyme against the heat-induced changes, indicating that the oxygen binding site plays a role in stabilizing the CuA site against structural perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependence of the electron spin-lattice relaxation rate from pulsed EPR of CUA and heme a in cytochrome c oxidase

This work shows the feasibility of using pulsed, saturation recovery EPR to study directly the magnetic relaxation properties of metal centers in cytochrome c oxidase in the 1.5-20 K range. Heme a and CuA both showed remarkably similar Tn temperature dependences in their spin-lattice relaxation rates. Either both are in environments with very similar protein backbone configurations (Stapleton, H.J., J.P. Allen, C.P. Flynn, D.G. Stinson, and S.R. Kurtz, 1980, Phys. Rev. Lett., 45:1456-1459; Allen, J.P., J.T. Colvin, D.G. Stinson, C.P. Flynn, and H.J. Stapleton, 1982, Biophys. J., 38:299-310), or the CuA is relaxed by nearby heme a. Spin-lattice relaxation of the nitrosylferrocytochrome a3 center in mixed valence oxidase showed enhancement of relaxation by a nearby paramagnetic center, most likely heme a.     

An electron nuclear double resonance investigation of redox-induced electronic structural change at CuA2+ in cytochrome c oxidase

We measured an electronic change at cysteine ligand(s) of the CuA2+ center brought on by reduction of other metal centers within cytochrome c oxidase, notably cytochrome a. This change specifically manifested itself as a modification in magnetic hyperfine coupling to the beta-protons of the beta-carbons adjacent to the cysteine sulfur in the CuA2+ coordination sphere. The electron nuclear double resonance ENDOR signals of these beta-protons had previously been assigned through study of selectively deuterated yeast oxidase. In the present study the ENDOR signals of the CuA2+ center were compared from the following forms of oxidase: resting (a3+.CuA2+.a3+3.CuB2+); mixed valence, 2-electron-reduced CO-ligated oxidase (a3+.CuA2+.a2+3CO.CuB+), and a more completely reduced mixed-valence CO-ligated oxidase. In agreement with previous studies on 3-electron-reduced oxidase, the latter more completely reduced oxidase showed cytochrome a preferentially reduced with respect to CuA, implying that the majority of paramagnetic CuA2+ centers had reduced cytochrome a partners. The ENDOR-resolved splitting of the beta-proton hyperfine features substantially decreased in going from the first two more oxidized forms to the more fully reduced latter form. Thus, the electronic structure of the CuA2+ center specifically monitored by hyperfine couplings to cysteine protons changed in response to a reductive event elsewhere in the protein. This structural change may correlate with the anticooperative redox interaction recently reported between cytochrome a and CuA.     

Extended X-ray absorption fine structure of copper in CuA-depleted, p-(hydroxymercuri)benzoate-modified, and native cytochrome c oxidase

Cytochrome c oxidase contains four redox-active metal centers: two heme irons, cytochromes a and a3, and two copper ions, CuA and CuB. Due to the paucity of spectroscopic signatures for both copper sites in cytochrome c oxidase, the ligands and structures for these sites have remained ambiguous. The specific depletion of CuA from the p-(hydroxymercuri)benzoate- (pHMB-) modified cytochrome c oxidase recently reported [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972] is herein described. Characterization of this enzyme shows that the structures of the remaining metal centers are essentially unperturbed by the CuA modification and depletion (P. M. Li, J. Gelles, and S. I. Chan, unpublished results). Copper extended X-ray absorption fine structure (EXAFS) measurements on the CuA-depleted cytochrome c oxidase reveal coordination of three (N, O) ligands and one (S, Cl) ligand at the CuB site. Comparison of EXAFS results obtained for the CuA-depleted, pHMB-modified, and "unmodified control" enzymes has allowed the deconvolution of the EXAFS in terms of the inner coordination spheres for CuA as well as CuB. On the basis of these data, it is found that the structure for the CuA site is consistent with two (N, O) ligands and two S ligands.     

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.     

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.     

Some properties of Nitrosomonas europaea cytochrome c oxidase (aa3-type) which lacks CuA

From Nitrosomonas europaea which had been cultivated in a medium deficient in copper, cytochrome c oxidase (aa3-type) which did not have CuA was purified. The oxidase did not show the 830-nm peak and its ESR spectrum differed greatly from that of the normal enzyme, which has two copper atoms, CuA and CuB, per molecule. However, the oxidase which did not have CuA showed almost the same cytochrome c oxidizing activity as the normal oxidase.     

The nature of CuA in cytochrome c oxidase

Kroneck et al. [(1988) FEBS Lett. 242, 70-74] have recently suggested, on the basis of a comparison with the EPR properties of nitrous oxide reductase, that cytochrome c oxidase contains a mixed-valence binuclear copper site, and that this is responsible for the EPR spectrum generally ascribed to CuA. Here we question this hypothesis in view of a multitude of analytical and spectroscopic data available. We maintain that a mononuclear Cu site with two cysteine sulfur and two imidazole nitrogen atoms as ligands is consistent with the current experimental information on the CuA site.     

Effect of high pH on the spectral and catalytic properties of beef heart cytochrome oxidase

Incubation of cytochrome oxidase at high pH induces changes in several spectral properties. The optical Soret maximum shifts to longer wavelength, and there is an apparent loss in intensity of the 655-nm band, effects that are normally assigned either to a spin-state transition in cytochrome a3 or to a reduction of heme a. However, magnetic circular dichroism spectra show that cytochrome a3 remains high spin and that both cytochrome a and cytochrome a3 are oxidized. At the same time, there is the appearance of a low-spin signal indicative of hydroxide-imidazole coordination which we assign as arising from a structural transition at cytochrome a, rather than at cytochrome a3, as has been proposed previously. With longer incubation times, a new copper signal appears with electron paramagnetic resonance parameters markedly different from those obtained from copper centers which have undergone denaturation. Spin quantitation establishes that this new resonance does not arise from CuA and suggests that high pH breaks the magnetic coupling present at the cytochrome a3-CuB center. A significant proportion of cytochrome a3 may be converted to a low-spin thiolate during this process.     

Evidence for a band III analogue in the near-infrared absorption spectra of cytochrome c oxidase.

Ground state near-infrared absorption spectra of fully reduced unliganded and fully reduced CO (a2+ CuA+ a3(2+)-CO CuB+) cytochrome c oxidase were investigated. Flash-photolysis time-resolved absorption difference spectra of the mixed-valence (a3+ CuA2+ a3(2+)-CO CuB+) and the fully reduced CO complexes were also studied. A band near 785 nm (epsilon approximately 50 M-1cm-1) was observed in the fully reduced unliganded enzyme and the CO photoproducts. The time-resolved 785 nm band disappeared on the same timescale (t1/2 approximately 7 ms) as CO recombined with cytochrome a3(2+). This band, which is attributed to the unliganded five coordinate ferrous cytochrome a3(2+), has some characteristics of band III in deoxy-hemoglobin and deoxy-myoglobin. A second band was observed at approximately 710 nm (epsilon approximately 80 M-1cm-1) in the fully reduced unliganded and the fully reduced CO complexes. This band, which we assign to the low spin ferrous cytochrome a, appears to be affected by the ligation state at the cytochrome a3(2+) site.     

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen.

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen is measured by transient absorption spectroscopy. The oxygen reaction is initiated by photolytic removal of CO from cytochrome oxidase, using a flash-pumped dye laser. The subsequent reaction of the cytochrome c-cytochrome oxidase complex with oxygen is reported at 550, 605, 744, and 830 nm at different cytochrome c:cytochrome oxidase ratios and different oxygen concentrations. In the absence of cytochrome c the time course of the reaction of the oxidase is well described by a triple exponential process at any of the measured wavelengths. The three processes are well resolved at high O2 levels (i.e. greater than 200 microM), where they reach first-order rate limits of 2.4 x 10(4), 7.5 x 10(3), and 650 s-1. When cytochrome c is added the oxidation of cytochrome a and one of the redox active cooper centers (CuA) are interrupted. The maximal effect of cytochrome c on the oxidation of the oxidase occurs at a c:aa3 ratio of 1. Cytochrome c reacts in a biphasic process with rates of up to 7 x 10(3) and 550 s-1 at high oxygen. The fast phase takes up 60% of the process, and this is independent of the cytochrome c:cytochrome oxidase ratio. The results are discussed in the context of a model in which electron entry into cytochrome oxidase from cytochrome c is via CuA, and cytochrome a functions to mediate electron transfer from CuA to the oxygen binding site. The role of CuA as initial electron acceptor in cytochrome c oxidase is related to its physical proximity to cytochrome c is the cytochrome c-cytochrome oxidase complex.     

Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.     

Solvent proton relaxation studies of cytochrome c oxidase solutions

The interaction of solvent water protons with the bound paramagnetic metal ions of beef heart cytochrome c oxidase has been examined. The observed proton relaxation rates of enzyme solutions had a negative temperature dependence, indicating a rapid exchange between solvent protons in the coordination sphere of the metal ions and bulk solvent. An analysis of the dependence of the proton relaxation rate on the observation frequency indicated that the correlation time, which modulates the interaction between solvent protons and the unpaired electrons on the metal ions, is due to the electron spin relaxation time of the heme irons of cytochrome c oxidase. This means that at least one of the hemes is exposed to solvent. The proton relaxation rate of the oxidized enzyme was found to be sensitive to changes in ionic strength and to changes in the spin states of the metal ions. Heme a3 was found to be relatively inaccessible to bulk solvent. Partial reduction of the enzyme caused a slight increase in the relaxation rate, which may be due to a change in the antiferromagnetic coupling between two of the bound paramagnetic centers. Further reduction resulted in a decreased relaxation rate, and the fully reduced enzyme was no longer sensitive to changes in ionic strength. The binding of cytochrome c to cytochrome c oxidase had little effect on the proton relaxation rates of oxidized cytochrome oxidase indicating that cytochrome c binding has little effect on solvent accessibility to the metal ion sites.     

Electronic state of heme in cytochrome oxidase III. The magnetic susceptibility of beef heart cytochrome oxidase and some of its derivatives from 7-200 K. Direct evidence for an antiferromagnetically coupled Fe (III)/Cu (II) pair.

The temperature dependence of the paramagnetic susceptibility of cytochrome oxidase and some of its derivatives has been measured from 7 to 200 K. The results obtained for the fully oxidized (resting) enzyme correspond exactly to the requirements of the model recently proposed by Palmer et al. (Palmer, G., Babcock, G. T., and Vickery, L. E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210) in which the enzyme possesses two magnetically isolated spin S = 1/2 centers and a spin-coupled S = 2 center. The S = 2 center paramagnetism has been interpreted as arising from a [cytochrome a33+(S = 5/2)--Cuu2+(S = 1/2)] antiferromagnetically coupled iron.copper binuclear complex of total spin S = 2 with -J greater than or equal to 200 cm-1. In addition, the wide temperature range used in the present studies has permitted an analysis of present and other available data (T less than 4K measurements) which readily accommodates results from this and other laboratories (Moss, T.H., Shapiro, E., King, T.E., Beinert, H., and Hartzell, C. R. (1978) J. Biol. Chem 253, 8072-8073) so that a fully consistent picture of the magnetic centers in cytochrome oxidase now appears to be available. Furthermore, anomalous magnetic behavior for the oxidized enzyme.cyanide complex has been interpreted in terms of an antiferromagnetic exchange interaction operating in the binuclear complex [cytochrome a33+.CN-(S = 1/2)--Cuu2+(S = 1/2)] with -J congruent to 40 cm-1. A structural model for the [cytochrome a3(3+)-bridge-CUu2+] center is advanced in which an imidazolate ion serves as the bridging ligand in a manner similar to that found in superoxide dismutase.     

Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria.

We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are much closer to those predicted from nonturnover measurements. In presence of uncouplers, Fea is always more reduced than Fec, but in the absence of uncouplers, the two centers track together. Unlike the uninhibited, coupled system, the redox behavior here is consistent with the known effect of the electrical membrane potential on electron distribution in the enzyme. Interestingly, in these circumstances (azide and uncoupler present), Fea behaves as if it were no longer the kinetically controlling electron donor to the bimetallic center.     

Electron spin echo studies of cytochrome c oxidase

We have studied the linear electric field effect in pulsed EPR of the "EPR-detectable copper" signal of beef heart cytochrome c oxidase and have compared our results with those for a variety of square planar and tetrahedral Cu(II) model compounds and with Cu(II) proteins containing either type 1 or type 2 copper. The electric field induced g shifts (linear electric field effect) for cytochrome oxidase are comparable in magnitude to those for simple Cu(II) complexes and for some copper proteins containing type 2 sites. The shifts are smaller than those for tetrahedral copper complexes and for type 1 copper sites. However, the magnetic field dependence of the linear electric field effect does not resemble that observed for any Cu(II) complex studied nor for type 1 copper. These findings cannot be reconciled with the tetrahedral Cu(II) model proposed by Greenaway, Chan, and Vincow ((1977) Biochim. Biophys. Acta 490, 62-78, 1977) to explain the unusual EPR spectrum of cytochrome oxidase.     

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.