Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance raman study

Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process.     

Conformational transitions and vibronic couplings in acid ferricytochrome c: a resonance Raman study.

Resonance Raman spectral changes in ferricytochrome c as a function of pH between 6.7 and 1.0 are reported and the structural implication is discussed in terms of the "core-expansion" model advanced by L. D. Spaulding et al. [(1975) J. Am. Chem. Soc. 97, 2517]. The data are interpreted as indicating the iron in high-spin ferricytochrome c (at pH 2.0) with two water molecules as axial ligands lies in the plane of the porphyrin ring. At pH 1.0 there is a different high-spin form of cytochrome c which has an estimated iron out-of-plane distance of approximately 0.46 A. The effect of a monovalent anion at pH 2.0 is to produce a thermal spin mixture with predominant low-spin species. Excitation at approximately 620 nm in acid cytochrome c (pH 2.0) enhances only three depolarized ring vibrations at 1623, 1555, and 764 cm-1. Marked enhancement of depolarized modes relative to polarized and anomalously polarized modes is attributed to the vibronic coupling between porphyrin pi leads to pi and porphyrin pi leads to iron (dpi) charge-transfer states.     

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.     

Radical formation in cytochrome c oxidase

The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O(2) redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O(2) with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77K at reaction times ranging from 50μs to 6ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO(2)(-) ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O(2) with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H(2)O(2)) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O(2) and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a(3). This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".     

Cytochrome c and cytochrome c peroxidase complex as studied by resonance Raman spectroscopy.

Complex formation between ferricytochrome c peroxidase (CCP) and ferricytochrome c from yeast [cyt(Y)] and horse heart [cyt(H)] was studied by resonance Raman spectroscopy. On the basis of a detailed spectral analysis of the free proteins, it was possible to attribute changes in the spectra of the complexes to the individual proteins. At pH 7.0 both cyt(Y) and cyt(H) binding induces an increase in the six-coordinate low-spin configuration of CCP from 9% to 19% at the expense of the five-coordinate high-spin state, which drops from 84% to 74%. In the free and complexed state, CCP exhibits a constant fraction of the six-coordinate high-spin form (approximately 7%). In addition to affecting the coordination state, there is also a cyt-specific structural response of CCP to complexation. In the cyt(Y)-CCP complex, the peripheral vinyl and propionate substituents of CCP are more rigidly fixed in the protein matrix, whereas binding of cyt(H) only slightly perturbs the conformations of these side chains. The biological significance of the conformational changes in CCP are discussed. In contrast to CCP, there are no detectable structural changes in either cyt(Y) or cyt(H) upon complex formation.     

Resonance Raman studies of CuA-modified cytochrome c oxidase

Modification of the CuA site in mammalian cytochrome c oxidase has been used to elucidate the functional role of this center in the catalytic cycle of the enzyme. Both heat treatment in detergents and chemical modification by p-(hydroxymercuri)benzoate (pHMB) convert CuA to a lower potential type II center and effectively remove the site from the electron-transfer pathway during turnover. In this study, resonance Raman spectroscopy has been employed to investigate the effects of these CuA modifications on the heme active sites. The Raman data indicate some environmental perturbation of the heme a3 chromophore in the modified derivatives. Only pHMB modification and SB-12 heat treatment produced significant effects in the Raman spectra of the fully reduced enzyme. These perturbations are much less evident in the spectra obtained within 10 ns of CO photolysis from the fully reduced species of the modified enzymes. Transient Raman studies further indicate that the half-time for CO religation in the modified enzymes is quite similar to that of the native protein.     

Resonance Raman applications in investigations of cytochrome c oxidase

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 655nm exhibit a Raman band at 755cm(-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 Cu(B). Time-resolved RR spectroscopy of whole mitochondria identified a band at 571cm(-1) arising from the oxygenated intermediate at Δt=0.4, 0.6 and 1.4ms. Bands at 804 and 780cm(-1) of the P and F intermediates were observed at Δt=0.6 and 1.4ms, 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.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.     

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.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. I. Binding of cytochrome c to cardiolipin/phosphatidylcholine membranes in the absence of oxidase.

The mechanism of interaction between cytochrome c and a solid-supported planar phosphatidylcholine membrane containing varying amounts of cardiolipin (0-20 mol%) has been studied over a wide range of protein concentrations (0-450 microM) and ionic strength conditions (10-150 mM), by direct measurement of protein binding using surface plasmon resonance (SPR) spectroscopy. The results demonstrate that cytochrome c binds to such phospholipid membranes in two distinct phases characterized by very different (approximately one order of magnitude) affinity constants. The second phase is dependent upon the prior occurrence of the first binding process. Although the binding affinities for both modes of binding are highly sensitive to both the cardiolipin concentration and the ionic strength of the buffer solution, indicating that electrostatic forces are involved in these processes, binding cannot be reversed by salt addition or by dilution. Furthermore, the final saturation levels of adsorbed protein are independent of ionic strength and cardiolipin concentration. These observations suggest that binding involves more than a simple electrostatic interaction. Invariance in the shapes of the SPR spectra indicates that no major structural transitions occur in the proteolipid membrane due to cytochrome c binding, i.e., the bilayer character of the lipid phase appears to be preserved during these interactions. Based on these results, a model of the lipid membrane-cytochrome c interaction is proposed that involves varying degrees of protein unfolding and subsequent binding to the membrane interior via hydrophobic forces.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Conformational change and histidine control of heme chemistry in cytochrome c peroxidase: resonance Raman evidence from Leu-52 and Gly-181 mutants of cytochrome c peroxidase.

Resonance Raman (RR) spectra are reported for Fe(III), Fe(II), and Fe(II)CO forms of site-directed mutants of the cytochrome c peroxidase variant CCP(MI), cloned in Escherichia coli. The Fe(II) form is five-coordinate (5-c) and high-spin at low pH, but it is six-coordinate (6-c) and low-spin at high pH except when the distal His-52 residue is replaced with Leu, showing the sixth ligand to be the His-52 imidazole. Although the Leu-52 mutant stays 5-c, it does undergo an alkaline transition, as revealed by upshifts and broadening of bands assigned to vinyl C = C stretching (1620 cm-1) and C beta-vinyl bending (402 cm-1). Similar changes are seen for CCP(MI) and other mutants. Thus the alkaline transition induces a conformational change that affects the vinyl groups, probably through changes in their orientation, and that permits the His-52 imidazole to bind the Fe. The RR band arising from the stretching of the proximal Fe(II)-imidazole bond contains components at ca. 235 and 245 cm-1 for CCP(MI), which are believed to reflect a double well potential for the H-bond between the proximal His-175 imidazole and the Asp-235 carboxylate group. Loss of this H-bond by mutation of Asp-235 to Asn results in the loss of these two bands and their replacement by a single band at 205 cm-1. Although the Fe(II)-imidazole stretching mode cannot be observed in the 6-c alkaline form of the enzyme, the sixth ligand in the alkaline form of CCP(MI) is photolabile, and the status of the Fe(II)-imidazole bond can be determined in the resulting 5-c-photoproduct. For CCP(MI) at alkaline pH, the conformation change induces an increase in the 235/245-cm-1 ratio, reflecting a perturbation of the H-bond potential. In the His-52----Leu mutant, a 205-cm-1 band appears along with the 235/245-cm-1 doublet at alkaline pH, indicating partial loss of the proximal H-bond due to the distal alteration. The effect of mutations that perturb the H-bonding network that extends from the distal to the proximal side of the heme is more dramatic: at alkaline pH, His-181----Gly, Arg-48----Leu, and Trp-51----Phe mutants show an Fe(II)-imidazole stretching mode at 205 cm-1 exclusively, indicating complete loss of the proximal Asp-235-His-175 H-bond.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

A cytochrome $\text{c}$ - cytochrome $\text{c}$ oxidase complex containing 0.8–1.0 moles of cytochrome $\text{c}$ per mole of cytochrome $\text{c}$ oxidase (heme a + a₃) was isolated as described by Ferguson-Miller, S., Brautigan, D.L., and Margoliash E., J. Biol. Chem. $\text{251}$, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 Å bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome $\text{c}$ was cross-linked to subunit II of cytochrome $\text{c}$ oxidase. Other cross-linked products were formed involving different subunits of cytochrome $\text{c}$ oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. Experiments are also described using N,N′-bis(3-succinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 Å bridging bifunctional reagent was a pair containing II+VI.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. II. Binding of cytochrome c to oxidase-containing cardiolipin/phosphatidylcholine membranes.

Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, whereas the final saturation level of bound protein is independent of salt concentration and corresponds to approximately 53% of the total cco molecules present in the membrane. This suggests a 1:1 binding stoichiometry between the two proteins. The nonspecific binding component is governed by electrostatic interactions between cyt c and the membrane lipids and results in a partially ionic strength-reversible protein-membrane association. Thus, hydrophobic interactions between cyt c and the membrane, which are the predominant mode of binding in the absence of cco, are greatly suppressed. Both the amount of nonspecifically bound protein and the binding affinity can be varied over a broad range by changing the ionic strength and the extent of CL incorporation into the membrane. Under conditions approximating the physiological state in the mitochondrion (i.e., 20 mol% CL and medium ionic strength), 1-1.5 cyt c molecules are bound to the lipid phase per molecule of cco, with a dissociation constant of 0.1 microM. The possible physiological significance of these observations is discussed.     

Conformational substates of bovine heart cytochrome c oxidase: the modified Volpe-Caughey variant

Kinetic and equilibrium binding studies using optically correlated, quantitative electron paramagnetic resonance (EPR) spectroscopy are reported for the reaction of a modified Volpe-Caughey preparation of bovine heart cytochrome c oxidase with anionic (F-, CN-) and gaseous (NO) ligands. A fast phase of cyanide and fluoride ligation can be attributed to an EPR-silent conformer(s), while the slow and medium phases of cyanide binding are correlated with the g = 12 conformer(s). Using dioxygen or ferricyanide, it is possible to modulate reciprocally the relative amounts of these two species, that together account for at least 95% of the active-site conformers of the resting form of the enzyme.     

Ultraviolet resonance Raman spectra of cytochrome c conformational states

Ultraviolet resonance Raman (UV RR) spectra are reported for ferricytochrome c from tuna and horse heart at pH 1.6, 7, 10, and 13, representing distinct conformational states of the protein (states II, III, IV, and V, respectively). The spectra were obtained with pulsed laser excitation at 200 and 218 nm, via H2 Raman shifting the fourth harmonic output of a pulsed YAG laser. At these deep UV wavelengths, strong enhancement is observed for vibrational modes associated with tryptophan, tyrosine, and phenylalanine side chains and with the amide groups of the polypeptide backbone. The amide I peak frequency is consistent with a dominant contribution from alpha-helical regions, although a broad high-frequency tail reflects a variety of unordered conformations. The peak frequency is 12 cm-1 higher for cytochrome c from tuna than from horse, suggesting a less tightly wound structure, which is consistent with the lower denaturation temperature previously reported for the tuna protein. The amide I peak broadens when native protein (state III) is converted to the low- or high-pH forms (states II and IV), reflecting some disordering of the polypeptide chain, but the peak frequencies are unshifted, establishing that the alpha-helical segments are not completely unfolded in these states. Raising the pH to 13 (state V), however, does produce a frequency upshift, reflecting helix unfolding. The amide II and III frequencies are likewise consistent with a dominant alpha-helix contribution in the native proteins; they gain intensity, and amide III is shifted to a lower frequency, in states II and IV, consistent with partial disordering.(ABSTRACT TRUNCATED AT 250 WORDS)