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Ferredoxins are ubiquitous proteins with electron transfer activity involved in a variety of biological processes. In this work, we investigated the characteristics and function of Fdx1 from Sorangium cellulosum So ce56 by using a combination of bioinformatics and of biochemical/biophysical approaches. We were able to experimentally confirm a role of Fdx1 in the iron-sulfur cluster biosynthesis by in vitro reduction studies with cluster-loaded So ce56 IscU and by transfer studies of the cluster from the latter protein to apo-aconitase A. Moreover, we found that Fdx1 can replace mammalian adrenodoxin in supporting the activity of bovine CYP11A1. This makes S. cellulosum Fdx1 the first prokaryotic ferredoxin reported to functionally interact with this mammalian enzyme. Although the interaction with CYP11A1 is non-physiological, this is-to the best of our knowledge-the first study to experimentally prove the activity of a postulated ISC-type ferredoxin in both the ISC assembly and a cytochrome P450 system. This proves that a single ferredoxin can be structurally able to provide electrons to both cytochromes P450 and IscU and thus support different biochemical processes. Combining this finding with phylogenetic and evolutionary trace analyses led us to propose the evolution of eukaryotic mitochondrial P450-type ferredoxins and ISC-type ferredoxins from a common prokaryotic ISC-type ancestor., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1--the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 Å away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.

Heterologous expression in yeast of mCYP11A1 fusions with different topogenic signals of yeast mitochondrial proteins for artificial channeling to different translocases of the inner membrane was used to gain insight in the mechanism of its topogenesis in mitochondria. To ensure insertion of the CYP11A1 domain into the inner mitochondrial membrane during the process of translocation, topogenic sequences containing transmembrane segments of Bcs1p(1-83), DLD(1-72), and full-sized AAC protein were used when constructing modified forms of CYP11A1, and the Su9(1-112) addressing signal was included to stimulate membrane insertion of CYP11A1 after its translocation to the matrix. Alternatively, to promote slippage of the hybrid molecules into the matrix, the hybrid of mCYP11A1 with the precursor of steroidogenic mitochondria matrix protein adrenodoxin (preAd) was designed. The extra sequences used for intramitochondrial sorting of CYP11A1 apparently ensured predicted topology of hybrid molecules in yeast mitochondria. All of the addressing sequences, containing transmembrane domains, provided effective insertion of the hybrid proteins AAC-mCYP11A1, Bcs1p(1-83)-mCYP11A1, DLD(1-72)-mCYP11A1 and Su9(1-116)-mCYP11A1 into the inner membrane. preAd-mCYP11A1 hybrid molecules were shown to be translocated across the inner membrane and tightly associated with the membrane on its matrix side but not membrane inserted. Measuring specific activities of hybrid proteins in the mitochondrial fractions upon addition of Ad and AdR showed that the hybrids predetermined for cotranslocational insertion of CYP11A1 into the inner membrane were more active in the reaction of cholesterol side-chain cleavage than those destined for insertion on the matrix side of the IM, the Ad-mCYP11A1 hybrid demonstrating only residual enzyme activity. The data obtained reinforce the proposal that complete transfer of the polypeptide chain into the matrix is not a necessary stage in its topogenesis, but rather persistent interaction of the polypeptide chain with the membrane during the process of translocation is of importance for heme binding, folding and membrane insertion.

The single mitochondrial type I [2Fe-2S] ferredoxin of the fission yeast Schizosaccharomyces pombe is produced as the carboxy terminal part of the electron-transfer-protein 1 (etp1) and cleaved off during mitochondrial import [Biochemistry 41 (2002) 2311-2321]. The UV/Vis (UV-visible) spectrum of the purified recombinant ferredoxin domain (etp1(fd)) expressed in Escherichia coli is similar to those of bovine Adx in the oxidized as well as in the reduced state. EPR (electronic paramagnetic resonance) studies revealed a correctly incorporated iron-sulfur cluster of the axial type. The redox potential of this protein was determined to be -353 mV, which is considerably lower than that of adrenodoxin (Adx, -273 mV). Several lines of evidence indicate that the protein forms dimers under physiological and denaturating conditions. Interestingly, the fission yeast ferredoxin could be shown to be active as an electron carrier in heterologous redox systems. It is able to transfer electrons to horse heart cytochrome c and to bovine cytochromes P450(scc) (CYP11A1) and P450(11 beta) (CYP11B1), thereby receiving electrons from bovine NADPH-dependent Adx reductase. The kinetics of substrate conversion in the etp1(fd)-supported CYP11A1 and CYP11B1-dependent systems mediated was studied.

Cytochrome P450scc (P450 11A1) catalyzes the conversion of cholesterol to pregnenolone, the first step in overall steroid hormone biosynthesis in steroidogenic tissues. On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F-G loop of P450 11A1 and mutated amino acid residues in this region. Wild type and mutant P450s 11A1 were expressed in E. coli and compared with respect to subcellular distribution and turnover number. About 30% of the wild type P450 was found in the bacterial cytosol indicating loose association of the enzyme with the membrane. The N210S/V211M and L218R/F219Y replacements increased the fraction of P450 in the bacterial cytosol 1.5-1.7-fold and the latter mutant also showed an almost two-fold increase of the turnover number. These data indicate that the putative F-G loop is the site of attachment to the membrane in P450 11A1 and changes in the enzyme-membrane interactions may affect the rate of catalysis.

Cytochrome P450scc (CYP11A1) is a protein attached to the inner surface of the inner mitochondrial membrane that uses cholesterol from the membrane phase as its substrate for the first step in steroid hormone synthesis. We investigated the mechanism by which CYP11A1 interacts with the membrane. Hydrophobicity profiles of CYP11A1 and two other mitochondrial cytochromes P450, plus a model structure of CYP11A1 using CYP2C5 as template, suggest that CYP11A1 has a monotopic association with the membrane which may involve the A' helix and the F-G loop. Deletion of the A' helix reduced the proportion of expressed CYP11A1 associated with the bacterial membrane fraction, indicating a role for the A' helix in membrane binding. However, introduction of a cysteine residue in this helix at position 24 (L24C) and subsequent labelling with the fluorescent probe N'-(7-nitrobenz-2-oxal,3-diazol-4-yl)ethylenediamine (NBD) failed to show a membrane localisation. Cysteine mutagenesis and fluorescent labelling of other residues appearing on the distal surface of the CYP11A1 model revealed that V212C and L219C have enhanced fluorescence and a blue shift following association of the mutant CYP11A1 with phospholipid vesicles. This indicates that these residues, which are located in the F-G loop, become localised to a more hydrophobic environment following membrane binding. Analysis of the quenching of tryptophan residues in CYP11A1 by acrylamide indicates that at least one and probably two tryptophans are involved in membrane binding. We conclude that CYP11A1 has a monotopic association with the membrane that is mediated, at least in part, by the F-G loop region.

On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F and G helices of mitochondrial P450s 27A1 and 11A. We introduced substitutions at Phe-207, Ile-211, and Phe-215 within putative helix F and at Trp-235 and Tyr-238 within putative helix G in P450 27A1 and compared wild type and mutants with respect to catalytic activity, product pattern, substrate binding, formation of hydrogen peroxide, and interaction with redox partner. Results indicate that the mutated residues are important for delivery of the correctly oriented substrate to the P450 active site. The I211K and F215K mutations, for example, affected the regioselectivity of P450 27A1-dependent hydroxylation reactions and conferred the P450 capacity to cleave the C-C bond of the substrate during the catalytic cycle. Studies of P450 11A1 indicate that Phe-202 has functions similar to those of its counterpart in P450 27A1 (Phe-215). We propose that putative helices F and G form the sides of the substrate-access channel, thus providing the additional mechanism to control regioselectivity of hydroxylation in mitochondrial P450s.

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.

Rotational diffusion measurements using EPR and saturation transfer EPR were applied to analyze complex formation between the electron-transfer components of the mitochondrial steroid-hydroxylating cytochrome P450 systems (CYP11A1 and CYP11B1) in phosphatidylcholine/phosphatidylethanolamine/cardiolipin vesicles prepared by octyl glucoside dialysis/adsorption. Octyl glucoside reconstitution of P450SCC results in large vesicles, which have an advantage over small vesicles in that vesicle tumbling does not contribute to measured rotational diffusion rates. Immobilization of spin-labeled adrenodoxin by both P450SCC and adrenodoxin reductase indicates equimolar complexation between P450SCC and adrenodoxin as well as between adrenodoxin reductase and adrenodoxin. Combination of rotational diffusion and antibody cross-linking confirmed the complexation of adrenodoxin with P450SCC and for the first time provided direct evidence of a complex between P450SCC and P45011beta in the membrane. In contrast, no evidence was found for the existence of adrenodoxin reductase-P450SCC complexes or a ternary complex of all three proteins. Thus, these experiments confirm the shuttle mechanism of electron transfer to vesicle-reconstituted P450SCC and P45011beta.

The antigenic structure of cytochrome P450scc was investigated by immunochemical identification of the peptides formed by chymotryptic cleavage of the protein and separated by reversed-phase and cation-exchange HPLCs. These procedures resulted in isolation and structural characterization of four homogeneous immunoreactive peptides which corresponded to the sequences Asn236-Tyr241, Leu266-Leu272, Ala381-Phe388, and Ser390-Trp400. These peptides contained several positively charged residues which were previously shown to participate in electrostatic interactions with adrenodoxin. Our data indicate that the positively charged residues of cytochrome P450scc are involved in formation of antigenic sites, and the antigenic determinants of the protein molecule coincide or overlap with the regions of polypeptide chain responsible for the interaction of the heme protein with adrenodoxin.

Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.

Mitochondrial monooxygenase systems are involved in the biosynthesis of glucocorticoids, mineralocorticoids, bile acids, and 1,25-dihydroxyvitamin D. The reactions are catalyzed by specific P450 enzymes that receive reducing equivalents via NADPH-ferredoxin oxidoreductase (adrenodoxin reductase) and ferredoxin (adrenodoxin). Although the three-dimensional structures of the individual components have not yet been solved, methods of expressing recombinant forms of these enzymes in Escherichia coli have allowed the use of site-directed mutagenesis to investigate the roles of specific amino acids in protein binding interactions, electron transfer, and catalysis. These studies have identified key charged residues in NADPH-ferredoxin oxidoreductase, ferredoxin, and P450scc, which are involved in electrostatic interactions critical for recognition, high-affinity binding, and electron transfer. The finding that the binding sites on ferredoxin for NADPH-ferredoxin oxidoreductase and P450 show significant overlap supports the proposed function for ferredoxin as a mobile electron shuttle between the reductase and P450 enzymes and is consistent with ferredoxin's role in serving multiple P450 isoforms.

Mitochondrial and microsomal cytochromes P-450SCC and P-450LM2 in the ferric substrate-free and substrate-bound states were studied by resonance Raman spectroscopy. In the spectra of cytochrome P-450SCC two conformational states (A and B) were detected, each of them constituting an equilibrium between a six-coordinated low-spin and a high-spin form. Both the conformational and the spin equilibria are pH- and temperature-dependent, which is in line with previously published results [Lange, R., Larroque, C., & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73)]. On the basis of well-resolved resonance Raman spectra, measured at different pH and temperatures, these equilibria were analyzed quantitatively. Both low-spin configurations of A and B exhibit different band patterns in the spin state marker band region, indicating differences in the active-site structures. While in the high-spin configuration of state A the heme iron remains weakly bound by a sixth ligand, the high-spin form of state B is five-coordinated. Binding of cholesterol to cytochrome P-450SCC causes a significant population of the high-spin forms, particularly of state A (62%). On the other hand, binding of 22R-hydroxycholesterol to the substrate-free enzyme leaves the overall spin equilibrium largely unchanged, i.e., six-coordinated low spin (76% A and 24% B). In both substrate-bound complexes, interactions between the substrate and the heme lead to small but distinct differences in the resonance Raman spectra of the low-spin form of state A. In contrast to cytochrome P-450SCC, the resonance Raman spectra of microsomal cytochrome P-450LM2 provide no indications for multiple conformers at 22 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined in vitro degradation of mitochondrial proteins by ATP-dependent protease in bovine adrenal cortex. Purified ATP-dependent protease degraded at least five proteins in vitro in the presence of ATP and Triton X-100 using mitochondrial fraction as a substrate. Two of the degraded proteins were identified as P-450scc and adrenodoxin reductase by immunoblotting. No degradation of P-45011 beta or adrenodoxin was detected. Purified P-450scc and adrenodoxin reductase were also degraded. By analyzing degradation products of P-450scc we identified 49 peptides and 59 cleavage sites. The size of the products was between 3 and 18 amino acid residues. The protease preferentially cleaved the carboxy-side of Leu, Phe, Ala, Val, Met, and some other amino acids. Since the protease (a matrix enzyme) is accessible to P-450scc and adrenodoxin reductase localized on the matrix side of inner membrane, the present results suggest that the protease might participate in the turnover of these two proteins and some other mitochondrial proteins.

1. Bovine adrenocortical P450scc was resolved into several fractions by chromatography on AH-Sepharose 4B followed by gel filtration on Toyopearl HW55S. All fractions contained P450scc of the same molecular size and the P450scc could be resolved into 3-4 major and more than 10 minor isoelectric point forms by isoelectric focusing on polyacrylamide gel in the presence of Emulgen 913. 2. Both the AH-Sepharose chromatography profile and the isoelectric focusing pattern of the adrenocortical P450scc were more complex than those of the corpus luteum P450scc. The corpus luteum P450scc was practically devoid of the neutral to acidic isoelectric point forms. 3. Three to four P450scc subfractions with different isoelectric focusing pattern were obtained from a purified preparation of adrenocortical P450scc by ion-exchange chromatography on DEAE-Toyopearl 650S or DEAE-Sephadex A25. These P450scc subfractions showed essentially the same spectral properties, catalytic activity, molecular weight and N-terminal amino acid sequence. 4. The most acidic (the latest eluting) subfraction was composed mostly of the neutral to acidic isoelectric point forms. The sedimentation characteristics of this subfraction was also studied. 5. The structural basis of the multiple molecular forms was discussed.