Inhibition of the cytochrome c/cytochrome c oxidase system by cytochrome c derivatives and related fragments

The oxidation of ferrocytochrome c mediated by cytochrome c oxidase was investigated in the presence of ferricytochrome c, trifluoroacetyl-cytochrome c, the heme fragments Hse65-[1-65] and Hse80-[1-80] and their respective porphyrin derivatives, as well as carboxymethylated apoprotein and related fragments, polycations, salts and neutral additives. The inhibition of the redox reaction by salts and neutral molecules, even if in theoretical agreement with their effect on electrostatic interactions, may alternatively be interpreted in terms of hydrophobicity. The latter can account for the inhibitory properties of trifluoroacetylated ferricytochrome c, similar to those of ferricytochrome c. On the assumption that the inhibitory properties of some of the investigated derivatives monitor their binding affinities to the cytochrome c oxidase at the cytochrome c binding sites, the experimental results do not confirm a primarily electrostatic character for the cytochrome c/cytochrome c oxidase association process. Strong indication was found that the cytochrome c C-terminal sequence is critically involved in the complex formation. Conformational studies by circular dichroism measurements and IR spectroscopy in solution and in solid state respectively, show that some of the derivatives examined may possibly acqkuire in the binding process to the oxidase, as secondary structure similar to that present in the native cytochrome c.     

Relation of the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin

The relationship between the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin was investigated. The rates of reduction of phosvitin-bound ferricytochrome c by cytochrome b2, ascorbate and the superoxide radical generated by xanthine oxidase wer repressed where the binding ratio was less than half the maximum, but at higher ratios they were restored gradually with increase in the ratio. The affinity of cytochrome b2 for cytochrome c was not affected by binding of cytochrome c to phosvitin. The redox potential of the bond form was lower than that of the free form and only decreased with decrease in the ratio. The conformatin around the heme moiety and the electronic structure of the heme group of bound ferricytochrome c were similar to those of free ferricytochrome c, but the conformational stability in the vicinity of the prosthetic group was related to the binding ratio as ratios above half the maximum and was well correlated with the reduction rate. Since the binding of cytochrome c to phosvitin is much stronger at binding ratios below half the maximum, these results suggest that this binding strength exclusively affects the conformational flexibility of the heme crevice in the cytochrome molecule, thus altering the reduction rate.     

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.     

Association of spin-labelled cardiolipin with dimyristoylphosphatidylcholine-substituted bovine heart cytochrome c oxidase. A generalized specificity increase rather than highly specific binding sites

The endogeneous lipid of bovine heart cytochrome c oxidase has been replaced by dimyristoylphosphatidylcholine using cholate-mediated exchange. The lipid-substituted preparation contained less than 1 mole cardiolipin per mole enzyme and possessed full oxidative activity. The association of spin-labelled cardiolipin with such lipid-substituted cytochrome oxidase preparations has been assayed using ESR spectroscopy. An average relative association constant 5.4-times that for phosphatidylcholine is obtained for cardiolipin. Measurements on preparations with increasing contents of unlabelled cardiolipin, introduced during lipid exchange, reveal that this selectivity corresponds to a generalized increase in specificity for all lipid association sites on the protein.     

Calorimetric studies of cytochrome oxidase-phospholipid interactions

Thermotropic phase transitions in phospholipid vesicles reconstituted with mitochondrial cytochrome oxidase (EC 1.9.3.1) were studied using differential scanning calorimetry. Both dimyristoylphosphatidylcholine (DMPC) and mixtures of DMPC and cardiolipin were used at different lipid-to-protein ratios. The incorporated protein reduces the energy absorbed during phase transitions of DMPC vesicles, and causes a small decrease in the transition temperature (tm). delta H depends on the amount of protein in the vesicles. This dependence indicates that about 72 DMPC molecules are influenced per cytochrome alpha alpha 3 monomer. The transition parameters remain unaffected by changes in ionic strength or by reduction of the enzyme. Incorporation of cytochrome oxidase depleted of subunit III into DMPC liposomes resulted in a larger decrease of tm, but the amount of perturbed phospholipids remains similar to that in the case of the intact enzyme. Incorporation of cytochrome oxidase into DMPC/cardiolipin vesicles counteracts the effect of cardiolipin in decreasing the enthalpy of the DMPC transition. Thus cytochrome oxidase segregates the phospholipids by attracting cardiolipin from the bulk lipid. Cytochrome c does not significantly affect this apparent cardiolipin 'shell' around membranous cytochrome oxidase.     

Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase

The lipid-soluble peroxides, tert-butyl hydroperoxide and peroxidized cardiolipin, each react with bovine cytochrome c oxidase and cause a loss of electron-transport activity. Coinciding with loss of activity is oxidation of Trp19 and Trp48 within subunits VIIc and IV, and partial dissociation of subunits VIa and VIIa. tert-Butyl hydroperoxide initiates these structural and functional changes of cytochrome c oxidase by three mechanisms: (1) radical generation at the binuclear center; (2) direct oxidation of Trp19 and Trp48; and (3) peroxidation of bound cardiolipin. All three mechanisms contribute to inactivation since blocking a single mechanism only partially prevents oxidative damage. The first mechanism is similar to that described for hydrogen peroxide [Biochemistry43:1003-1009; 2004], while the second and third mechanism are unique to organic hydroperoxides. Peroxidized cardiolipin inactivates cytochrome c oxidase in the absence of tert-butyl hydroperoxide and oxidizes the same tryptophans within the nuclear-encoded subunits. Peroxidized cardiolipin also inactivates cardiolipin-free cytochrome c oxidase rather than restoring full activity. Cardiolipin-free cytochrome c oxidase, although it does not contain cardiolipin, is still inactivated by tert-butyl hydroperoxide, indicating that the other oxidation products contribute to the inactivation of cytochrome c oxidase. We conclude that both peroxidized cardiolipin and tert-butyl hydroperoxide react with and triggers a cascade of structural alterations within cytochrome c oxidase. The summation of these events leads to cytochrome c oxidase inactivation.     

A quantitative test for superoxide radicals produced in biological systems.

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.     

The steady-state rate equation for cytochrome c oxidase based on a minimal kinetic scheme

A minimal catalytic cycle for cytochrome c oxidase has been suggested, and the steady-state kinetic equation for this mechanism has been derived. This equation has been used to simulate experimental data for the pH dependence of the steady-state kinetic parameters, kcat and Km. In the simulations the rate constants for binding and dissociation of cytochrome c and for two internal electron-transfer steps have been allowed to vary, whereas fixed experimental values (for pH 7.4) have been used for the other rate constants. The results show that the dissociation of the product, ferricytochrome c, cannot be rate-limiting under all conditions, but that intramolecular electron-transfer steps also limit the rate. They also demonstrate that Km can differ considerably from the dissociation constant for the cytochrome c-oxidase complex. Published values for the rate constant for the dissociation of ferricytochrome c are too small to account for the steady-state rates. It is suggested that, at high concentrations, ferryocytochrome c transfers an electron to a cytochrome c molecule which remains bound to the oxidase. This can also explain the nonhyperbolic kinetics, which is observed at low substrate concentrations.     

心磷脂引起细胞色素C的氧化

心磷脂—细胞色素C—细胞色素C氧化酶体系吸收光谱的研究发现:心磷脂与氧化态细胞色素C结合产生230nm吸收峰;心磷脂与还原态细胞色素C作用,230nm吸收值上升,550nm吸收值下降,表明心磷脂可以引起细胞色素C的氧化。     

Characterization of the interaction of cytochrome c and mitochondrial ubiquinol-cytochrome c reductase

Characterization of the steady state kinetics of reduction of horse ferricytochrome c by purified beef ubiquinol-cytochrome c reductase, employing 2,3-dimethoxy-5-methyl-6-decylbenzoquinol as reductant, has shown that: 1) the dependence of the reaction on quinol and on ferricytochrome c concentration is consistent with a ping-pong mechanism; 2) the pH optimum of the reaction is near 8.0; 3) the effect of ionic strength on the apparent Km and the TNmax of the reaction for the native cytochrome c is small, and at higher cytochrome c concentrations substrate inhibition is observed; 4) the effect of ionic strength on the kinetic parameters for the reaction of 4-carboxy-2,6-dinitrophenyllysine 27 horse cytochrome c is much larger than for the native protein; and 5) competitive product inhibition is also observed with a Ki consistent with the binding affinity of ferrocytochrome c for Complex III, as determined by gel filtration. In addition, direct binding measurements demonstrated that ferricytochrome c binds more tightly than the reduced protein to Complex III under low ionic strength conditions and that under these conditions more than one molecule of cytochrome c is bound per molecule of Complex III. Exchange of Complex III into a nonionic detergent decreases this excess nonspecific binding. Measurement of the rates of dissociation of the oxidized and reduced 1:1 complexes of cytochrome c and Complex III by stopped flow was consistent with the disparity of binding affinities, the dissociation rate constant for ferrocytochrome c being about 5-fold higher than that for the ferric protein. A model which accounts for the properties of this system is described, assuming that cytochrome c bound to noncatalytic sites on the respiratory complex decreases the catalytic site binding constant for the substrate.     

Kinetic and structural differences between cytochrome c oxidases from beef liver and heart

1. The cytochrome content of beef liver mitochondria differs from that of beef heart mitochondria by an eightfold lower cytochrome aa3 and a twofold lower cytochrome b and c + c1 content. 2. The kinetic properties of cytochrome c oxidases from beef liver and heart were measured with intact cytochrome c-depleted membranes, deoxycholate-dissolved membranes, and with the isolated enzymes at various cytochrome c concentrations with an oxygen electrode. Under all conditions a higher V was found for the liver enzyme, both for the low-affinity and for the high-affinity binding site for cytochrome c. Differences were also found for the Km of the two enzymes. 3. Isolated beef heart mitochondria contained about twice as much cardiolipin than beef liver mitochondria. The isolated enzymes contained one mole cardiolipin per mole of the heart enzyme, but 2 moles cardiolipin per mole of the liver enzyme. 4. By application of a high performance sodium dodecylsulfate gel electrophoretic system the two isolated enzymes could be separated into 13 different protein components, three of which (polypeptides VIa, VIIa and VIII) were found to differ in their apparent molecular weights. The functional meaning of cytochrome c oxidase isoenzymes in liver and heart is discussed.     

Cytochrome c mediates electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase by free diffusion along the surface of the membrane.

Ubiquinol oxidase can be reconstituted from ubiquinol-cytochrome c reductase (Complex III) and cytochrome c oxidase (Complex IV) whose endogenous phosphatidylcholine and phosphatidylethanolamine have been replaced by dimyristoylglycerophosphocholine. Phase transition of the lipid has no effect on Complex III and Complex IV activities assayed separately, but ubiquinol oxidase activity rapidly decreases as the temperature is lowered through the phase transition. A spin-labelled yeast cytochrome c derivative has been synthesized. Binding of the cytochrome c to liposomes demonstrates that only cardiolipin is involved under the conditions used for the ubiquinol oxidase experiments. In liposomes consisting of cardiolipin and dimyristoylglycerophosphocholine, e.s.r. (electron-spin-resonance) measurements show that rotational diffusion of cytochrome c is slowed in the gel phase of the latter lipid. We propose that the cytochrome c pool is bound to cardiolipin molecules, whose lateral and rotational diffusion in the bilayer is adequate to account for electron-transport rates.     

Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN*

The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (<r.m.s. deviation>_bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (<r.m.s. deviation>_bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.     

Membrane binding induces destabilization of cytochrome c structure.

The effect of membranes binding on the structure and stability of ferricytochrome c was studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Association of cytochrome c with phospholipid membranes containing phosphatidylglycerol as a model acidic phospholipid results in only slight, if any, perturbation of the protein secondary structure. However, upon membrane binding, there is a considerable increase in the accessibility of protein backbone amide groups to hydrogen-deuterium exchange, which suggests a lipid-mediated loosening and/or destabilization of the protein tertiary structure. A lipid-induced conformational perturbation of ferricytochrome c is also indicated by a marked decrease in the thermodynamic stability of the membrane-bound protein. Upon binding to membranes containing dimyristoylphosphatidylglycerol (DMPG) or dioleoylphosphatidylglycerol (DOPG) as a single lipid component, the denaturation temperature of ferricytochrome c decreases by approximately 30 degrees C. This is accompanied by a decrease in the calorimetric enthalpy of denaturation, particularly for the DMPG-associated protein. With ferricytochrome c bound to membranes containing a mixture of DMPG (or DOPG) and zwitterionic phosphatidylcholine, the extent of structural perturbation depends on the surface density of the negatively charged lipid head groups, becoming smaller with decreasing proportions of acidic phospholipid in the membrane. The observed destabilization of protein structure mediated by acidic phospholipids (and possibly formation of folding intermediates at the membrane surface) may represent a general property of a larger class of water-soluble proteins for which membrane binding is governed by electrostatic forces.     

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.     

Cytochrome c oxidase inhibition by N-retinyl-N-retinylidene ethanolamine, a compound suspected to cause age-related macula degeneration

The endogenous lipophilic and cationic compound N-retinyl-N-retinylidene ethanolamine (A2E) is suspected to cause age-related macula degeneration. It inhibits cytochrome c oxidase, detaches proapoptotic proteins from mitochondria, and induces apoptosis in mammalian retinal pigment epithelial cells (M. Suter, C. E. Remé, C. Grimm, A. Wenzel, M. J??ttela, P. Esser, N. Kociok, M. Leist, and C. Richter, 2000, J. Biol. Chem. 275, 39625-39630). The inhibition of cytochrome c oxidase is highly specific for A2E and is observed with the solubilized and reconstituted enzyme. In the dark, inhibition is overcome by cardiolipin or other acidic phospholipids. With illumination, inhibition is stronger, becomes complete with prolonged exposure, and is then no longer abrogated by cardiolipin. Cardiolipin effectively displaces A2E from cytochrome c oxidase, suggesting noncovalent binding of A2E to the enzyme. We conclude that A2E is a potent cytochrome c oxidase-specific inhibitor which interferes with the binding of cytochrome c to cytochrome c oxidase and, in the light, causes persistent modifications of the enzyme.     

Equilibrium thermodynamics of a physiologically-relevant heme-protein complex

We used isothermal titration calorimetry to study the equilibrium thermodynamics for formation of the physiologically-relevant redox protein complex between yeast ferricytochrome c and yeast ferricytochrome c peroxidase. A 1:1 binding stoichiometry was observed, and the binding free energies agree with results from other techniques. The binding is either enthalpy- or entropy-driven depending on the conditions, and the heat capacity change upon binding is negative. Increasing the ionic strength destabilizes the complex, and both the binding enthalpy and entropy increase. Increasing the temperature stabilizes the complex, indicating a positive van't Hoff binding enthalpy, yet the calorimetric binding enthalpy is negative (-1.4 to -6.2 kcal mol(-)(1)). We suggest that this discrepancy is caused by solvent reorganization in an intermediate state. The measured enthalpy and heat capacity changes are in reasonable agreement with the values estimated from the surface area change upon complex formation. These results are compared to those for formation of the horse ferricytochrome c/yeast ferricytochrome c peroxidase complex. The results suggest that the crystal and solution structures for the yeast complex are the same, while the crystal and solution structures for horse cytochrome c/yeast cytochrome c peroxidase are different.     

The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome c oxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate-respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome c oxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS-induced loss of cytochrome c oxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome c oxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS-induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

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.