Sterol 14alpha-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.     

A new technique for assaying cytochrome P450 enzyme activity in a single cell

A new microspectrofluorometric technique for measuring the ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 (CYP)1A1 in single living cells is described. The system, which uses a perfusion chamber and an HPLC pump, allowed cells to be stained, fixed, blocked, and washed by injecting each treatment solution into the on-line carrier stream of buffer from the sampling block of the HPLC pump. After addition of the substrate 7-ethoxyresorufin, the fluorescence intensity of the metabolite resorufin was measured in individual cells. Fluorescence intensity steeply increased to a unique peak for each cell and then decreased to the basal level. Furthermore, CYP1A1 in each cell was stained with its antibody and quantified using the fluorescence intensity of an FITC-conjugated secondary antibody. EROD activity was normalized using the FITC fluorescence. The results show that the initial slopes and peak values of resorufin production by the cells were dependent on the CYP1A1 level. Treatment of hepatocytes with two nonspecific P450 inhibitors, cimetidine and SKF-525A, suppressed EROD activity.     

A novel molecular modelling study of inhibitors of the 17alpha-hydroxylase component of the enzyme system 17alpha-hydroxylase/17,20-lyase (P-450(17alpha)).

The enzyme 17alpha-hydroxylase/17,20-lyase (P-450(17alpha) has recently become the focus of research into the fight against hormone dependent prostate cancer. However, the specific nature of this enzyme, in particular, the dual role of its active site, remains unknown. In our drive to elucidate further information regarding P-450(17alpha), and in light of our experience of other cytochrome P-450 enzymes, we chose to consider each part of this complex enzyme separately (i.e. the 17alpha-hydroxylase (17alpha-OHase) and the 17,20-lyase components). We therefore initiated a series of molecular modelling studies involving the construction of a 'substrate heme complex' for each of the two components. Here, we consider the construction and use of the complex for the 17alpha-OHase component of this enzyme. Using this approach, we have successfully considered: the binding of steroidal and non-steroidal reversible inhibitors: the structural features necessary for potent inhibition: and, rationalised the mode of action of a number of compounds whose inhibitory activity has not been previously explained, for example aminoglutethimide (an inhibitor of another related cytochrome P-450 enzyme, aromatase AR). The study concludes that the ability of the inhibitors of 17alpha-OHase to undergo polar polar interaction with the active site and for the compounds to closely mimic the substrate plane is a major factor in determining potency. Factors such as log P (log of the partition coefficient value for the distribution of a compound between octanol and water) would then appear to determine the extent of overall inhibitory activity. Overall, the study suggests that the novel substrate-heme complex approach has provided a good approximation of the 17alpha-OHase active site and has proved to be a useful tool in drug design and discovery.     

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Delta(5)P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450(C17)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(C17)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(C17)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including 17OH-Delta(5)P, 17OH-P, DHEA and Delta(4). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450(C17) inhibitor. This is the first detailed immunohistochemical mapping of P450(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.     

Liver cytochrome P450 3A endoplasmic reticulum-associated degradation: a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol

The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.     

A three-dimensional model of aromatase cytochrome P450.

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.     

A structural and data-driven approach to engineering a plant cytochrome P450 enzyme

Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s. However, the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450 s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87 D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.     

Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme

Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage lambda gt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83, 8064-8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8-2 [Cullin, C. & Pompon, D. (1988) Gene 65, 203-217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous-CO spectrum typical of cytochrome P-450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4-oxidation, quinidine 3-hydroxylation and N-oxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH-cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH-cytochrome P-450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.     

P450(camr), a cytochrome P450 catalysing the stereospecific 6- endo-hydroxylation of (1 R)-(+)-camphor

Rhodococcus sp. NCIMB 9784 accumulated 6-endo-hydroxycamphor 3 when grown on (1R)-(+)-camphor 1 as sole carbon source. The structure of 3 has been unambiguously assigned for the first time using X-ray crystallography. A soluble cytochrome P450 hydroxylase, induced by growth on (1R)-(+)-camphor and designated P450_camr, has been isolated from the bacterium Rhodococcus sp. NCIMB 9784. Using authentic 6-endo hydroxycamphor as standard, a cell-free system consisting of pure P450_camr and putidaredoxin and putidaredoxin reductase from Pseudomonas putida confirmed that the enzyme hydroxylates (1R)-(+)-camphor specifically in the 6-endo position, in contrast to the 5-exo hydroxylation catalysed by the well-studied P450_cam from P. putida. P450_camr has a molecular mass of approximately 44 kDa, and a pI of 4.8. Electronic Publication     

The interaction of cytochrome P450 17alpha with NADPH-cytochrome P450 reductase, investigated using chemical modification and MALDI-TOF mass spectrometry

The lysine residues of guinea pig P450 17alpha were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17alpha prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17alpha was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17alpha in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17alpha with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.     

Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.     

17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, a selective mechanism-based inhibitor of cytochrome P450(17 alpha) (steroid 17 alpha-hydroxylase/C17-20 lyase)

A new compound, 17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, MDL 27,302, has been designed and synthesized as a mechanism-based inhibitor of cytochrome P450(17 alpha). The time-dependent inactivation of human testicular P450(17 alpha) is irreversible by dialysis and requires the cofactor, NADPH; Kiapp. 90 nM (determined on cynomolgous monkey testis enzyme). Inactivation was not affected by the nucleophile DTT, suggesting retention of the inhibitor in the enzyme active site during the inactivation process. Inhibition is specific to the cyclopropylamino compound, since the isopropylamino- and cyclobutylamino-analogs were not inhibitory. Enzymatic specificity of MDL 27,302 for P450(17 alpha) was demonstrated by its failure to inhibit steroid 21-hydroxylase and the cholesterol side chain cleavage enzyme (P450scc). Both the 17 alpha-hydroxylase and C17-20 lyase activities of cytochrome P450(17 alpha) of human testis microsomes were inhibited by MDL 27,302.     

EpoK, a cytochrome P450 involved in biosynthesis of the anticancer agents epothilones A and B. Substrate-mediated rescue of a P450 enzyme

The epothilones are a new class of highly promising anticancer agents with a mode of action akin to that of paclitaxel but with distinct advantages over that drug. The principal natural compounds are epothilones A and B, which have an epoxide in the macrocyclic lactone ring, and C and D, which have a double bond instead of the epoxide group. The epoxidation of epothilones C and D to A and B, respectively, is mediated by EpoK, a cytochrome P450 enzyme encoded in the epothilone gene cluster. Here we report high-yield expression of EpoK, characterization of the protein, demonstration that the natural substrate can prevent-and even reverse-denaturation of the protein, identification of ligands and surrogate substrates, development of a high-throughput fluorescence activity assay based on the H(2)O(2)-dependent oxidation of 7-ethoxy-4-trifluoromethylcoumarin, and identification of effective inhibitors of the enzyme. These results will facilitate improvements in the yields of epothilones C and D and the engineering of EpoK to prepare novel epothilone analogues. Furthermore, the finding that the denatured enzyme is rescued by the substrate offers a potential paradigm for control of the P450 catalytic function.     

人类细胞色素P450与药物氧化代谢遗传多态性分子机制的研究现状

近年,在表型及基因型上均发现存在药物氧化代谢多态性,特别是对于人类细胞色素Ｐ４５０氧化酶与药氧化代谢遗传多态性的关系进行了深入的研究。有关ＣＹＰ２Ｄ６、ＣＹＰ２Ｃ１９等的突变已大多被鉴定;ＣＹＰ１Ａ１、ＣＹＰ１Ａ２等在表型存在多态性而确切的遗传机制尚不清楚。     

Membrane topology of guinea pig cytochrome P450 17 alpha revealed by a combination of chemical modifications and mass spectrometry

Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH-cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17alpha was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17alpha was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 17alpha and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17alpha's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N- and C-terminal domains and the putative F-G loop were concluded to be in or near the membrane-binding domains of P450 17alpha.     

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.     

Ontogenesis of rabbit liver cytochrome P450. Evidence for a cytochrome P450-IIE (3a)-related form prevailing during the post-natal period

The liver hydroxylating system, mainly composed of cytochromes P450, is not highly active during foetal life. If develops after birth and reaches the adult level several weeks post-partum. We have studied the ontogenesis of rabbit cytochrome P450 during the post-natal period. Total P450 as well as isozymes 2, 3b, 3c, 4 and 6 were measured. The evolution of these proteins with ageing, together with qualitative modification of an electrophoretic profile, produced evidence of an early developing P450 prevailing from one week to three weeks after birth. We isolated and characterized a cytochrome, called P450 2y, from two-week liver microsomes. It is closely related to P450 3a, an adult form of rabbit P450 induced by ethanol. They have similar molecular masses, the same lambda max of CO-reduced spectrum and exhibit immunological cross-reactivity. However, we cannot conclude that the two proteins are identical from N-terminal amino acid analysis or the two-dimensional gel electrophoresis pattern. These results, as well as the recent evidence of two different genes coding for the P450 3a family, strengthen the idea that P450 2y and 3a are distinct proteins. P450 2y seems to be an early developing form abundant soon after birth, while P450 3a is a delayed form appearing like most P450 isozymes during the fourth post-natal week. Besides the quantitative development during perinatal life, there is an important qualitative modification of liver cytochrome P450 content.     

Screening of potential substrates or inhibitors of cytochrome P450 17A1 (CYP17A1) by electrochemical methods

The electrochemical redox reactions of the recombinant form of human cytochrome P450 17A1 (CYP17A1) were investigated. The hemoprotein was immobilized on the electrode modified with a biocompatible nanocomposite material based on the membrane-like synthetic surfactant didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of DDAB/Au/CYP17A1 electrodes were investigated using cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Analysis of electrochemical behavior of cytochrome P450 17A1 was performed in the presence of a natural substrate, pregnenolone (1), known inhibitor, ketoconazole (2), and in the presence of synthetic derivatives of pregnenolone: acetyl pregnenolone (3), cyclopregnenolone (4), and tetrabromo-pregnenolone (5). Ketoconazole, the azole inhibitor of cytochromes P450, blocked catalytic current in the presence of the substrate, pregnenolone (1). Compounds 3–5 did not demonstrate substrate properties towards the electrode/CYP17A1 system. Compound 3 did not influence catalytic activity with pregnenolone, whereas compounds 4 and 5 demonstrated some inhibitory activity. Thus, electrochemical redox reactions of CYP17A1 may serve as an adequate substitution of the reconstituted system, which requires additional redox partners for manifestation of catalytic activity of hemoproteins of the cytochrome P450 superfamily.     

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.