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Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b 5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (k cat / K m) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b 5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that –as in human CYP17A1–these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b 5 protein in the lyase reactions. Oxidation of steroids by 17α-hydroxylation and lyase reaction via mouse Cyp17a1. [Display omitted] • Recombinant mouse cytochrome P450 17A1 (Cyp17a1) enzyme was expressed and purified. • The catalytic parameters of mouse Cyp17a1 was analyzed with relevant steroids • Mouse Cyp17a1 had much higher catalytic efficiency than human CYP17A1. • The enzyme catalytic efficiency of mouse Cyp17a1 was significantly stimulated by b 5 protein. [ABSTRACT FROM AUTHOR]

Dissociation constants Kd for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b5 (b5) stimulates metabolism of this substrate and dramatically decreases the substrate-related Kd values. Experiments are performed to test if the b5-mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b5 (Mn b5) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b5 stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b5 is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b5, with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450. [Copyright &y& Elsevier]

The metabolism of selected substrates by cytochromes P450 (P450) 2B1, 2B4, and 2B6 was compared, and the effects of cytochrome b5 (b5) on these reactions were assessed. There did not appear to be any trends regarding the effects of b5 when the metabolism of a given substrate by the different P450 enzymes was compared. The changes in spin states of the P450 enzymes as a result of interactions with substrates and cytochrome b5 were also determined. Only P450 2B4 demonstrated a relationship between spin state, reaction coupling and b5 effects. The rates of benzphetamine and 7-ethoxy-4-trifluoromethylcoumarin metabolism by the three enzymes could be correlated with the proportions of high spin heme. Similarly, the proportion of reaction coupling during the metabolism of selected substrates was approximately equal to the proportion of high spin P450. The data are interpreted to indicate that a P450 conformational equilibrium coordinately regulates catalysis and spin state changes. [Copyright &y& Elsevier]

We have compared experimental measurements and Brownian dynamic calculations for the reduction of oxidised adult human haemoglobin by reduced bovine cytochrome b5 over a range of ionic strengths. Our calculations suggest that the presence of molecular electrostatic fields have a significant role to play in the formation of the electron transfer complexes. These results predict that electron transfer occurs within an ensemble of similarly weakly docked complexes, the formation of which is strongly ionic strength dependent. Application of electron tunneling analysis to the complexes allows us to predict the rates of electron transfer within each ensemble of complexes as a function of ionic strength. The outcome of this theoretical study is compared with experimental rate measurements. A comparison of the results obtained from adult and embryonic haemoglobins, at a fixed ionic strength, indicates a significant difference in the characteristics of complex formation. These data emphasise the role played by electrostatic interactions in this important physiological reaction. [ABSTRACT FROM AUTHOR]