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 A 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.

https://www.pnas.org/content/pnas/108/25/10139.full.pdf

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system.

Cytochrome P450 mechanisms continue to surprise and delight, although the field is growing to maturity and the completely unexpected is less frequently encountered. Experimentally, the past few years have seen major progress in characterizing the intermediates that are formed as molecular oxygen is activated to the final oxidizing species. All the intermediates, with the exception of the critical ferryl species, have now been directly observed by various spectroscopic and crystallographic methods. The ferric peroxo anion has been found to act as the oxidizing agent with a growing range of highly electrophilic substrates. In contrast, the proposed role for the ferric hydroperoxo complex as an electrophilic oxidizing agent remains a matter of debate, as the evidence advanced in support of the proposal is circumstantial and contradictory. Although the ferryl species remains elusive, it is increasingly clear that it plays the predominant role as the oxidizing agent in the P450 catalytic cycle. A second area that has recently received considerable attention is the mechanism of hydrocarbon hydroxylation, the key question being whether the radical rebound mechanism that has held sway for three decades is in fact valid. The contradictory results obtained with radical and cation probes, which have provided most of the new evidence, must be resolved by further experimentation in order for this quest ionto be settled. The development of a two-state model for the catalytic action of P450 enzymes may be one of the most important recent advances in the field, as it provides a ready explanation for a variety of otherwise contradictory data, some of which argues for concerted and some for nonconcerted oxidation mechanisms. No doubt, the next few years will uncover novel aspects of P450 function and will lead to deeper and more sophisticated understanding of the catalytic mechanisms of the amazing family of P450 enzymes.

Substrate Oxidation by Cytochrome P450 Enzymes

Publisher Summary Congenital adrenal hyperplasia (CAH) is a family of disorders of adrenal steroidogenesis resulting from an inherited deficiency of one of several enzymes necessary for normal steroid synthesis. This chapter describes the recent advances in congenital adrenal hyperplasia. These advances have come from the interdisciplinary collaboration of embryologists, teratologists, enzymologists, immunogeneticists, population geneticists, steroid endocrinologists, and gender psychologists. The chapter describes asimplified scheme of adrenal steroidogenesis. It provides an overview of the additional evidence that supports the concept that the adrenal fasciculate and glomerulosa function as two separate glands. This has been gained from the recent study of patients with 21-hydroxylase deficiency. New data shows that in CAH, there is a 21-hydroxylation defect in the zona fasciculata of simple virilizers and salt wasters, whereas zona glomerulosa is defective in salt wasters and not in simple virilizers. The data suggest that there is one enzyme involved in the 21-hydroxylation of the 17-hydroxy and 17-deoxy pathways of adrenal steroidogenesis in zona fasciculata.

An Update of Congenital Adrenal Hyperplasia

Male pseudohermaphroditism: the complexities of male phenotypic development.

Publisher Summary This chapter discusses the aspects of ovarian cholesterol metabolism that appear to be subject to control, directly or indirectly, by gonadotropins. These include lipoprotein uptake, de novo sterol synthesis, intracellular cholesterol transport, mitochondrial cholesterol side chain cleavage, and the synthesis and hydrolysis of cholesteryl esters. The chapter focuses on the corpus luteum as this is the best studied component of the ovary with respect to sterol metabolism. Luteal cells have a unique life cycle; their steroidogenic activity and, therefore, their cholesterol requirements change dramatically as they differentiate from granulosa cells and undergo a period of secretory activity followed by a phase of waning function and structural demise.

Cholesterol metabolism by ovarian tissue.

https://www.jbc.org/content/247/5/1462.full.pdf

Studies on the Biosynthetic Conversion of Cholesterol into Pregnenolone SIDE CHAIN CLEAVAGE OF A t-BUTYL ANALOG OF 20α-HYDROXYCHOLESTEROL, (20R)-20-t-BUTYL-5PREGNENE-3β,20-DIOL, A COMPOUND COMPLETELY SUBSTITUTED AT C-22

Studies on the Biosynthetic Conversion of Cholesterol into Pregnenolone: SIDE CHAIN CLEAVAGE OF SOME 20-p-TOLYL ANALOGS OF CHOLESTEROL AND 20α-HYDROXYCHOLESTEROL

Fragments formed by the side chain cleavage of a 20-aryl analog of 20alpha-hydroxycholesterol by adrenal mitochondria.

Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol.

Patrick D. McDonald

Papers

Studies on the Biosynthetic Conversion of Cholesterol into Pregnenolone SIDE CHAIN CLEAVAGE OF A t-BUTYL ANALOG OF 20α-HYDROXYCHOLESTEROL, (20R)-20-t-BUTYL-5PREGNENE-3β,20-DIOL, A COMPOUND COMPLETELY SUBSTITUTED AT C-22

Studies on the Biosynthetic Conversion of Cholesterol into Pregnenolone: SIDE CHAIN CLEAVAGE OF SOME 20-p-TOLYL ANALOGS OF CHOLESTEROL AND 20α-HYDROXYCHOLESTEROL

Fragments formed by the side chain cleavage of a 20-aryl analog of 20alpha-hydroxycholesterol by adrenal mitochondria.

Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol.