The Interaction of Bovine Adrenodoxin with CYP11A1 (Cytochrome P450scc) and CYP11B1 (Cytochrome P45011β)

Abstract

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1⅐CO and CYP11B1⅐CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4 -108), Adx-(4 -114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/ S112W, and Y82S/S112W (the last four mutants are ⌬113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.Cytochromes P450 (CYP11A1, CYP11B1, and CYP11B2) 1 of the inner mitochondrial membrane catalyze various hydroxylation steps in the biosynthesis of steroid hormones (mineralocorticoids, glucocorticoids, and androgens) in vertebrates (1, 2). The electrons required for these hydroxylation reactions are provided by NADPH and are transferred to the cytochromes P450, which mediate the final oxygen activation, via a small electron transport chain. Comparable with the components of the soluble bacterial cytochrome P450 systems, such as P450 cam (CYP101) and P450 terp (CYP108), the proteins of this electron transport system are an FAD-containing NAD(P)H-dependent reductase and an iron-sulfur protein of the [2Fe-2S] ferredoxin type.Adrenodoxin, the ferredoxin of the adrenal gland (3), has been studied intensively by chemical modifications (4 -6) and later by a great number of site-directed mutagenesis studies (7-13). Recently crystal structures of a truncated form, Adx-(4 -108) (14), and of full-length Adx (15) have been solved. In contrast to the bacterial systems, the reductase and the cytochromes of the mitochondrial systems are membrane-associated or membrane-bound proteins, respectively (1, 16).The reduction kinetics of the soluble bacterial cytochrome P450 cam is well understood (17-20), whereas little is known about the kinetics of the membrane-bound mitochondrial cytochromes P450 (21-23). Nevertheless previous investigations point to considerable differences in the reaction mechanisms and in the regulation in these two types of cytochrome P450 systems.To get deeper insight into the mechanism causing these differenc...

Stripping Down the Mitochondrial Cholesterol Hydroxylase System, a Kinetics Study

“…These reduction rates show the same tendency of deceleration than the observed k cat /K m values. The k cat /K m values reported here at 100 mM KCl being 0.0034 s Ϫ1 M Ϫ1 are in excellent agreement with previously published results of our group (19). Due to the fact that it is only possible to measure the first reduction step of the heme iron of CYP11A1 using the stopped flow technique, we cannot directly link the apparent rate constants to the k cat values of the CYP11A1-dependent cholesterol conversion.…”

“…In this mixing mode, the traces of reduction of CYP11A1 by Adx were also best described by a mono-exponential equation. These measurements are in good agreement with previous observations using this mixing mode (19). The pronounced dependence on the ionic strength of CYP11A1 reduction is shown in Fig.…”

“…The system was made anaerobic as described previously (19). The reaction buffer consisted of 50 mM HEPES (pH 7.3), varying amounts of potassium chloride, and 0.05% Tween 20.…”

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

“…Since the other Adx species seem to be capable as well of binding to the region around Lys-193, it may be postulated that the complex configuration involving Lys-193 may require a reorientation of the Adx species before ET is enabled, but that phosphorylated Adx may not require reorientation as has been proposed for other Adx species. 37 This assumption may provide an explanation for the observation that WT-Adx and the two Adx mutants preferentially bind to the region near residue Lys-405 and additionally provides an explanation for the biexpo-nential behavior of the phosphorylated species. Since phospho-rylated Adx displays biexponential electron-transfer kinetics, we suggest that the phosphorylation of T71 increases the efficiency of the electron-transfer pathway of this second complex configuration.…”

“…[37][38][39][40] Prior to mixing, syringe A contained CYP11A1 (2 μM) while syringe B was filled with NADPH (200 μM), AdR (2 μM), and varying concentrations of the different Adx species in the range from 0.5 to 64 μM.…”