Identification of the binding site on cytochrome c1 for cytochrome c

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.     

Ferredoxin electron transfer site on cytochrome c3. Structural hypothesis of an intramolecular electron transfer pathway within a tetra-heme cytochrome.

To specify electron exchanges involving Desulfovibrio desulfuricans Norway tetra-heme cytochrome c3, the chemical modification of arginine 73 residue, was performed. Biochemical and biophysical studies have shown that the modified cytochrome retains its ability to both interact and act as an electron carrier with its redox partners, ferredoxin and hydrogenase. Moreover, the chemical modification effects on the cytochrome c3 1H NMR spectrum were similar to that induced by the presence of ferredoxin. This suggests that arginine 73 is localized on the cytochrome c3 ferredoxin interacting site. The identification of heme 4, the closest heme to arginine 73, as the ferredoxin interacting heme helps us to hypothesize about the role of the three other hemes in the molecule. A structural hypothesis for an intramolecular electron transfer pathway, involving hemes 4, 3 and 1, is proposed on the basis of the crystal structures of D. vulgaris Miyazaki and D. desulfuricans Norway cytochromes c3. The unique role of some structural features (alpha helix, aromatic residues) intervening between the heme groups, is proposed.     

Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze approximately 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide cross-links, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide cross-links, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.     

Preliminary 1H-NMR studies of the interaction between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway

The complex formation of two electron transfer proteins, cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway, has been shown by 1H-NMR spectroscopy. Presence of ferredoxin I produces ferricytochrome c3 1H-NMR spectrum modifications. The chemical shift of perturbated heme methyl resonances has been used to determine the stoichiometry of the complex. At pH 7.6 and 20 degrees C, the two proteins were found to form a complex 1:1 with an association constant, KA, of 10(4) M-1. Two of the four hemes are affected by presence of ferredoxin I and may be involved in the electron transfer sites. The heme methyl resonances are average resonances of free and bound cytochrome c3 resonances, indicating a fast exchange process on the NMR time scale.     

Use of specific lysine modifications to locate the reaction site of cytochrome c with cytochrome oxidase.

The reaction of cytochrome c with trifluoromethylphenyl isocyanate was carried out under conditions which led to the modification of a small number of the 19 lysines. Extensive ion-exchange chromatography was used to separate and purify six different derivatives, each modified at a single lysine residue, lysines 8, 13, 27, 72, 79, and 100, respectively. The only modifications which affected the activity of cytochrome c with cytochrome oxidase (EC 1.9.3.1) were those of lysines immediately surrounding the heme crevice, lysines 13, 27, 72, and 79, and also lysine 8 at the top of the heme crevice. In each case, the modified cytochrome c had the same maximum velocity as that of native cytochrome c, but an increased Michaelis constant for high affinity phase of the reaction. This supports the hypothesis that the cytochrome oxidase reaction site is located in the heme crevice region, and the highly conserved lysine residues surrounding the heme crevice are important in the binding.     

The reaction domain on Rhodospirillum rubrum cytochrome c2 and horse cytochrome c for the Rhodospirillum rubrum cytochrome bc1 complex

The interaction of the Rhodospirillum rubrum cytochrome bc1 complex with R. rubrum cytochrome c2 and horse cytochrome c was studied using specific lysine modification and ionic strength dependence methods. In order to define the reaction domain on cytochrome c2, several fractions consisting of mixtures of singly labeled carboxydintrophenyl-cytochrome c2 derivatives were employed. Fraction A consisted of a mixture of derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2, while fractions C1, C2, C3, and C4 were mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The rate of the reaction of fraction A was found to be nearly the same as that of native cytochrome c2. However, the rate constants of fractions C1-C4 were found to be more than 20-fold smaller than that of native cytochrome c2. These results indicate that lysine residues surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. Since the same domain is involved in the reaction with the photosynthetic reaction center, cytochrome c2 must undergo some type of rotational or translational diffusion during electron transport in R. rubrum. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse cytochrome c also involves the heme crevice domain.     

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

Structure of the cytochrome b6f complex: quinone analogue inhibitors as ligands of heme cn

A native structure of the cytochrome b(6)f complex with improved resolution was obtained from crystals of the complex grown in the presence of divalent cadmium. Two Cd(2+) binding sites with different occupancy were determined: (i) a higher affinity site, Cd1, which bridges His143 of cytochrome f and the acidic residue, Glu75, of cyt b(6); in addition, Cd1 is coordinated by 1-2 H(2)O or 1-2 Cl(-); (ii) a second site, Cd2, of lower affinity for which three identified ligands are Asp58 (subunit IV), Glu3 (PetG subunit) and Glu4 (PetM subunit). Binding sites of quinone analogue inhibitors were sought to map the pathway of transfer of the lipophilic quinone across the b(6)f complex and to define the function of the novel heme c(n). Two sites were found for the chromone ring of the tridecyl-stigmatellin (TDS) quinone analogue inhibitor, one near the p-side [2Fe-2S] cluster. A second TDS site was found on the n-side of the complex facing the quinone exchange cavity as an axial ligand of heme c(n). A similar binding site proximal to heme c(n) was found for the n-side inhibitor, NQNO. Binding of these inhibitors required their addition to the complex before lipid used to facilitate crystallization. The similar binding of NQNO and TDS as axial ligands to heme c(n) implies that this heme utilizes plastoquinone as a natural ligand, thus defining an electron transfer complex consisting of hemes b(n), c(n), and PQ, and the pathway of n-side reduction of the PQ pool. The NQNO binding site explains several effects associated with its inhibitory action: the negative shift in heme c(n) midpoint potential, the increased amplitude of light-induced heme b(n) reduction, and an altered EPR spectrum attributed to interaction between hemes c(n) and b(n). A decreased extent of heme c(n) reduction by reduced ferredoxin in the presence of NQNO allows observation of the heme c(n) Soret band in a chemical difference spectrum.     

Amino acid sequences of the two heme c-binding sites of Pseudomonas cytochrome-c peroxidase

The amino acid sequences of the two heme c-containing tryptic peptides of Pseudomonas cytochrome-c peroxidase have been determined. The tryptic peptides were isolated from two cyanogen bromide fragments of the protein. Both heme-binding sites have the Cys-X-Y-Cys-His structure characteristic of c-type cytochromes. The sequences of the two peptides show distinct homology with each other, suggesting the occurrence of gene doubling during evolution of the protein molecule. The function of the heme c moieties in the catalytic cycle of the enzyme is discussed on the basis of their homology with the proximal histidine region of peroxidase (horseradish peroxidase and yeast cytochrome-c peroxidase) and cytochromes (horse cytochrome c and Pseudomonas cytochrome c-551).     

Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies.

Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.     

The interaction of ferredoxin and glutamate synthase: cross-linking and immunological studies

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.     

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy.

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.     

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes.     

Spectroscopic analysis of the interaction between cytochrome c and cytochrome c oxidase

Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.     

Cross-linked amino acids in the protein pair S13-S19 and sequence analysis of protein S13 of Bacillus stearothermophilus ribosomes

The 30S ribosomal subunits from Bacillus stearothermophilus were cross-linked under native conditions with the bifunctional reagent diepoxybutane. The dominant protein-protein cross-link in the 30S ribosomal subunit between proteins S13 and S19 [Brockm?ller, J., & Kamp, R.M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935] was isolated on a preparative scale. The presence of a single cross-link site between cysteine-83 of protein S13 and histidine-68 of protein S19 was established by microsequence analysis of isolated cross-linked peptides. This cross-link site was further confirmed by different analytical methods including fast atom bombardment mass spectrometry of the cross-linked peptide. The cross-linking site is located in the highly conserved C-terminal regions of proteins S13 and S19. In addition, the complete amino acid sequence of protein S13 from B. stearothermophilus is determined. Sequence comparison with the homologous Escherichia coli protein S13 revealed 58% identical amino acid residues.     

A covalently linked complex of cytochrome P-450 with adrenodoxin. Localization of the adrenodoxin binding site of cytochrome P-450

Cytochrome P-450scc and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of a cross-linked complex and 6% of free cytochrome P-450scc was obtained after purification on cholate-Sepharose. Cytochrome P-450scc in the cross-linked complex is not reduced in the presence of NADPH and adrenodoxin reductase, but completely preserves its high spin form in the presence of Tween-20 or pregnenolone. The use of radioactive labelled adrenodoxin, chemical cleavage of cytochrome P-450scc from the cross-linked complex by o-iodosobenzoic acid and HPLC for separation of peptides demonstrated that the cytochrome P-450scc complex with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450scc, i.e., Leu 88-Trp108 and Leu368-Trp417.