Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c.

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state.     

Mobility of fluorescent derivatives of cytochrome c in mitochondria

Motion of cytochrome c bound to giant (2-10-micron diam) mitochondria isolated from the waterbug Lethocerus indicus was examined using the technique of fluorescence recovery after photobleaching. Fluorescent cytochrome c was exchanged for native cytochrome c through partly damaged outer membrane. Recovery profiles were not statistically different when the fluorescence from iron-free cytochrome c or fluorescein-labeled cytochrome c was used and were essentially the same in the presence or absence of an uncoupler. In the presence of excess porphyrin cytochrome c, the apparent diffusion coefficient was 6 X 10(-11) cm2/s in 0.3 M sucrose-mannitol-EDTA and 3 X 10(-10) cm2/s in 0.10 M KCl/0.10 M sucrose. At concentrations of porphyrin cytochrome c that are stoichiometric with cytochrome c oxidase and for mitochondria in which excess cytochrome c was washed away, two components were observed in the recovery profile. The diffusion coefficient of the fast component was 1 X 10(-10) cm2/s. The second component showed no recovery during the time scale of measurement (D less than 10(-12) cm2/s). We speculate on the origin of the immobile fraction.     

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase.

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.     

Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c.

Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.     

Biosynthesis of cytochrome c oxidase by isolated rat liver mitochondria.

When isolated mitochondria which have been labeled with [3H]leucine are solubilized and treated with anti-serum specific for cytochrome c oxidase, labeled polypeptides which correspond to the three largest polypeptides of this enzyme are immunoprecipitated. This indicates that the three largest polypeptides of cytochrome c oxidase which have Mr of 66,000, 39,000, and 23,000 are synthesized by isolated mitochondria whereas the three smallest ones which have Mr of 14,000, 12,500, and 10,000 are not. The smallest polypeptides are probably synthesized on cytoplasmic ribosomes as has been demonstrated in other systems by in vivo studies. These results are the first demonstration that isolated mammalian mitochondria are capable of synthesizing some of their own polypeptide components. The antiserum used in this study was prepared to highly purified cytochrome c oxidase (12.4 nmol of heme a + a3/mg of protein) from rat liver mitochondria. This antiserum gives a single precipitin line when tested by the Ouchterlony double diffusion technique. Its specificity has been demonstrated by the fact that it: 1) only precipitates heme a + a3, not hemes b, c, or c1, when added to solubilized mitochondria, 2) inhibits cytochrome c oxidase activity at least 85%, and 3) precipitates only those polypeptides found in purified cytochrome c oxidase when added to solubilized mitochondria labeled in vivo.     

Structure and orientation of cytochrome c oxidase in crystalline membranes. Studies by electron microscopy and by labeling with subunit-specific antibodies.

The structure and the orientation of cytochrome c oxidase molecules in crystalline cytochrome c oxidase membranes (Vanderkooi, G., Senior, A.E., Capaldi, R.A., and Hayashi, H. (1972) Biochim. Biophys. Acta 274, 38-48) were studied by image analysis of electron micrographs and by reacting the crystalline preparations with immune gamma-globulins against individual cytochrome c oxidase subunits. Binding of gamma-globulins to the membranes was detected by the following two methods: (a) electrophoretic identification of gamma-globulin polypeptides in the washed membranes; (b) electron microscopic examination of the negatively stained membranes. The membranes bound immune gamma-globulins against subunit IV (which faces the matrix side in intact mitochondria) but failed to bind immune gamma-globulins against subunits II + III (which face the outer side of the inner membrane in intact mitochondria). In contrast, solubilized cytochrome c oxidase bound either of the two immune gamma-globulins. All cytochrome c oxidase molecules in the crystalline membranes are thus asymmetrically arranged so that subunit IV faces outward and subunits II + III face toward the interior. This orientation is opposite to that found with intact mitochondria. The data also suggest that the crystalline membranes form closed vesicles which are impermeable to externally added gamma-globulins.     

The nature of zinc in cytochrome c oxidase.

The zinc ion in bovine heart cytochrome c oxidase can be completely depleted from the enzyme with mercuric chloride without denaturing the protein. The metal atom stoichiometry of 5Cu/4Fe/0Zn/2Mg obtained for the enzyme following HgCl2 treatment indicates that this depletion is highly selective. Zinc depletion exposes one cysteine on subunit VIa and one cysteine on subunit VIb for N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine (1,5-I-AEDANS) labelling, suggesting that the zinc plays a structural role in the protein by providing a bridge between these two subunits. Although the treatment of cytochrome c oxidase with mercuric chloride inhibits the steady-state activity of the enzyme, subsequent removal of the Hg2+ bound to cysteine residues by 1,5-I-AEDANS significantly reverses the inhibition. This latter result indicates that the removal of the zinc itself does not alter the steady-state activity of the enzyme.     

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake.

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N',N'-tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c. 2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa3 by ferrocyanide. 3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake. 4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.     

Hydrophobic interactions of cytochrome c oxidase. Application to the purification of the enzyme from rat liver mitochondria.

The binding of rat liver cytochrome c oxidase to phenyl-Sepharose and various alkyl and omega-aminoalkyl agarose gels has been studied. Deoxycholate-solubilized cytochrome c oxidase was tightly bound to hexyl, octyl, omega-aminohexyl, omega-aminooctyl agarose as well as to phenyl-Sepharose. This hydrophobic interaction was used for the purification of cytochrome c oxidase. The enzyme which was eluted from phenyl-Sepharose was devoid of NADH (NADPH)-acceptor reductase activities. The heme a content was 15.4 nmol per mg of protein. The purified enzyme was resolved into seven polypeptides upon polyacrylamide gel electrophoresis in sodium dodecylsulfate with molecular weights of 40,000, 23,200, 21,500, 14,500, 12,600, 8900, and 4900. Antibodies raised in rabbits against the pure enzyme did not cross-react with cytochrome c oxidases from either beef heart or yeast mitochondria. Cytochrome c oxidase bound to octyl-Sepharose or phenyl-Sepharose exhibited a very low catalytic activity. The possible modes of interaction of cytochrome c oxidase with the hydrophobic ligands are discussed.     

Determination of the orientation distribution of adsorbed fluorophores using TIRF. II. Measurements on porphyrin and cytochrome c.

The theory for determination of the orientation of adsorbed fluorescent molecules using total internal reflection fluorescence, as explained in part I of this series, is illustrated by measurements on adsorbed tetramethylpyridinium porphyrin (H2TMPyP) and porphyrin cytochrome c molecules. The results are encouraging, although for porphyrin cytochrome c the scatter in the obtained order parameters is substantial. For H2TMPyP molecules adsorbed on glass the orientation distribution depends on the solution concentration. At low concentration, the H2TMPyP molecules are more or less randomly oriented, whereas at high concentrations a broad distribution around an angle of 46 degrees between the porphyrin plane and surface was found. For cytochrome c adsorbed on glass and indium tin oxide it was impossible to interpret the data in terms of orientation distributions because of the scatter in the results. The total fluorescence as a function of the polarization angle psi of the incident light beam corresponds to an average angle between the porphyrin group and the surface of 30 degrees-40 degrees. Despite the strong electric dipole moment of the protein, the orientation distribution seems to be independent on the (imposed) electrical potential of the interface.     

Dicyclohexylcarbodiimide binds specifically and covalently to cytochrome c oxidase while inhibiting its H+-translocating activity

We have investigated the covalent binding of dicyclohexylcarbodiimide (DCCD) to cytochrome c oxidase in relation to its inhibition of ferrocytochrome c-induced H+ translocation by the enzyme reconstituted in lipid vesicles. DCCD bound to the reconstituted oxidase in a time- and concentration-dependent manner which appeared to correlate with its inhibition of H+ translocation. In both reconstituted vesicles and intact beef heart mitochondria, the DCCD-binding site was located in subunit III of the oxidase. The apolar nature of DCCD and relatively minor effects of the hydrophilic carbodiimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, on H+ translocation by the oxidase indicate that the site of action of DCCD is hydrophobic. DCCD also bound to isolated cytochrome c oxidase, though in this case subunits III and IV were labeled. The maximal overall stoichiometries of DCCD molecules bound per cytochrome c oxidase molecule were 1 and 1.6 for the reconstituted and isolated enzymes, respectively. These findings point to subunit III of cytochrome c oxidase having an important role in H+ translocation by the enzyme and indicate that DCCD may prove a useful tool in elucidating the mechanism of H+ pumping.     

The oxidation of exogenous cytochrome c by mitochondria. Resolution of a long-standing controversy

Several reports in the past have dealt with the oxidation of cytochrome c added to suspensions of rat liver mitochondria. Yet, it is generally believed that the cytochrome cannot penetrate the outer membrane. Probably it has been assumed that the permeability of the outer membrane to cytochrome c is very low but finite, and that fast oxidation may be observed if time is allowed for sufficient penetration before initiation of electron flow. Here we show that this view is false. The main fraction of rat liver mitochondria, as isolated by conventional procedures, does not catalyse any significant oxidation of added cytochrome c, even after prolonged incubation. The observed appreciable oxidation of added cytochrome c is catalysed by a very small fraction (5-12%) of the mitochondria that apparently has a damaged outer membrane. Consequently, the turnover of cytochrome oxidase is very high in this fraction during oxidation of added cytochrome c. This finding readily explains why Moyle and Mitchell (e.g., FEBS Lett. 88 (1978) 268-272; 90 (1978) 361-365) have failed to observe proton translocation by cytochrome oxidase during oxidation of ferrocytochrome c added to rat liver mitochondria, which has been their main reason for rejecting the proton-pumping function of cytochrome oxidase.     

Electron paramagnetic resonance of the excited triplet state of metal-free and metal-substituted cytochrome c.

The photoactivated metastable triplate states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 10(-4), 342 x 10(-4) and 353 x 10(-4) cm-1, respectively, and are temperature invariant over the temperature ranges studied. AN E value at 4 K of 73 x 10(-4) cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrins derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the E value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2 > zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt c are interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-1 is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The magnitude of E value is approximately 60 x 10(-4) cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of > or = 40 cm-1 is estimated. In contrast to those for the metal cytochromes, the magnitude of E value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.     

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.     

Porphyrin cytochrome c. pH effects and interaction with cytochrome-c oxidase

1. Porphyrin cytochrome c, the iron-free derivative of cytochrome c, has been used extensively as a fluorescent analog of cytochrome c. It appears as though its fluorescence intensity but not its relative quantum yield is affected by pH in the physiological range; an apparent pK of about 6.2 is found suggesting a histidine close to the porphyrin. 2. The fluorescence intensity of the porphyrin cytochrome c in the presence of cytochrome c oxidase is independent of pH; this suggests that the oxidase has the capacity to control the pK of whichever group is responsible for the pH sensitivity of the free porphyrin cytochrome c. The most likely candidate for this pH-sensitive group is histidine-18. The N-3 nitrogen of this residue forms one of the axial ligands to the iron in the intact cytochrome c but it is uncoordinated in the iron-free derivative.     

An enzyme kinetics and 19F nuclear magnetic resonance study of selectively trifluoroacetylated cytochrome c derivatives.

The reaction of cytochrome c with ethyl thioltrifluoroacetate was carried out under conditions which led to the selective trifluoroacetylation of a small number of the 19 lysines. The mixture of derivatives was separated by ion-exchange chromatography and four different derivatives with well-resolved 19F nuclear magnetic resonance (NMR) spectra were obtained. Peptide mapping techniques indicated that one of these derivatives contained a single trifluoroacetyl group at lysine 22, and another derivative was singly labeled at lysine 25. The trifluoroacetylated lysine 22 derivative was fully active toward both succinate-cytochrome c reductase (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) white the trifluoroacetylated lysine 25 derivative was fully active toward the reductase, but had a threefold greater Michaelis constant in the cytochrome oxidase reactin. This supports the hypothesis that the cytochrome oxidase binding site is located in the heme cervice region, and that Lys-25 is important in the binding. 19FNMR spectra of the cytochrome c derivatives bound to phospholipid vesicles were obtained. The reasonably narrow line widths (35-65 Hz) and good sensitivity of the trifluoroacetyl resonances indicated that they might be useful probes for the interaction of cytochrome c with intact mitochondria.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.