Stripping Down the Mitochondrial Cholesterol Hydroxylase System, a Kinetics Study*

The origin of steroid hormones in mammals is cholesterol that is metabolized by the mitochondrial CYP11A1 system. The cytochrome P450 is fed with reduction equivalents via a small electron transfer chain consisting of NADPH, adrenodoxin reductase, and adrenodoxin. Though the redox behavior of the individual protein components has been studied previously, the kinetics of the system in its entirety has not yet been analyzed. In this study we combine surface plasmon resonance experiments to determine the binding constants for the different pairs of redox partners with measurements of the pre-steady-state kinetics of the different reaction steps of this system and steady-state kinetics. We could correlate the individual protein-protein interactions with the effect of distinct reduction-oxidation steps on the overall catalytic activity of the CYP11A1 system. For the first time, we were able to follow the reduction of each of the protein components of this system within one measurement when we mixed all oxidized protein components with NADPH. These measurements allowed the determination of the individual apparent rate constants for the reduction of all three proteins involved. In addition, variation of the ionic strength in these experiments revealed different optimum salt concentrations for the reduction of adrenodoxin reductase and adrenodoxin, respectively, and unraveled dramatically changing reduction rates of CYP11A1 by adrenodoxin.

• Publication year

  2004

• Venue

  Journal of Biological Chemistry

• Publication date

  2004-08-13

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1074/JBC.M402798200PMID 15181009
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Bacterial (CYP101) and mitochondrial P450 systems-how comparable are they?

  2003cited by this paper
• Deletions in the loop surrounding the iron-sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450(scc) (CYP11A1).

  2002cited by this paper
• The Loop Region Covering the Iron-Sulfur Cluster in Bovine Adrenodoxin Comprises a New Interaction Site for Redox Partners*

  2001cited by this paper
• The Interaction of Bovine Adrenodoxin with CYP11A1 (Cytochrome P450scc) and CYP11B1 (Cytochrome P45011β)

  2001cited by this paper
• Adrenodoxin: Structure, stability, and electron transfer properties

  2000cited by this paper
• Optical biosensor studies on the productive complex formation between the components of cytochrome P450scc dependent monooxygenase system

  1999cited by this paper
• Specific Aspects of Electron Transfer from Adrenodoxin to Cytochromes P450scc and P45011β*

  1997cited by this paper
• Molecular recognition and electron transfer in mitochondrial steroid hydroxylase systems.

  1997cited by this paper
• The kinetics of protein‐protein recognition

  1997cited by this paper
• Cytochrome P450: structure, function, and generation of reactive oxygen species.

  1996cited by this paper
• Mitochondrial specificity of the early steps in steroidogenesis.

  1995cited by this paper
• Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes. Identification by complementary site-specific mutations.

  1993cited by this paper
• Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization.

  1993cited by this paper
• Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding.

  1992cited by this paper
• Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 Fe-2S/ cluster ligands.

  1992cited by this paper
• Expression of mammalian P450s in Escherichia coli.

  1991cited by this paper
• Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding.

  1989cited by this paper
• Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin.

  1989cited by this paper
• Sensitive assay of cytochrome P450scc activity by high-performance liquid chromatography.

  1989cited by this paper
• Cloning and sequence analysis of adrenodoxin reductase cDNA from bovine adrenal cortex.

  1987cited by this paper
• The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc.

  1987cited by this paper
• Adrenodoxin interaction with adrenodoxin reductase and cytochrome P-450scc. Cross-linking of protein complexes and effects of adrenodoxin modification by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

  1984cited by this paper
• Steroidogenic electron transport in adrenal cortex mitochondria

  1982cited by this paper
• The kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin.

  1981cited by this paper
• Mitochondrial cytochrome P-450scc. Mechanism of electron transport by adrenodoxin.

  1980cited by this paper
• Adrenodoxin reductase . adrenodoxin complex. Rapid formation and breakdown of the complex and a slow conformational change in the flavoprotein.

  1980cited by this paper
• Adrenodoxin reductase.adrenodoxin complex. Flavin to iron-sulfur electron transfer as the rate-limiting step in the NADPH-cytochrome c reductase reaction.

  1979cited by this paper
• Ionic effects on adrenal steroidogenic electron transport. The role of adrenodoxin as an electron shuttle.

  1979cited by this paper
• Properties of an adrenal cytochrome P-450 (P-450SCC) for the side chain cleavage of cholesterol.

  1978cited by this paper
• Purification of adrenodoxin reductase, adrenodoxin, and cytochrome P-450 from adrenal cortex.

  1978cited by this paper
• Adrenodoxin reductase and adrenodoxin. Mechanisms of reduction of ferricyanide and cytochrome c.

  1977cited by this paper
• Formation of a cytochrome P-450scc-adrenodoxin complex.

  1977cited by this paper
• Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

  1976cited by this paper
• Adrenodoxin reductase-adrenodexin complex.

  1976cited by this paper
• Iron-Sulfur Proteins

  1975cited by this paper
• The stoichiometry of the conversion of cholesterol and hydroxycholesterols to pregnenolone (3beta-hydroxypregn-5-en-20-one) catalysed by adrenal cytochrome P-450.

  1974cited by this paper
• Studies on adrenal steroid hydroxylases. Complex formation of the hydroxylase components.

  1973cited by this paper
• Studies on adrenal steroid hydroxylases. Oxidation-reduction properties of adrenal iron-sulfur protein (adrenodoxin).

  1973cited by this paper
• Studies on adrenal steroid hydroxylases. Molecular and catalytic properties of adrenodoxin reductase (a flavoprotein).

  1973cited by this paper
• Components of the electron transport system in adrenal steroid hydroxylase. Isolation and properties of non-heme iron protein (adrenodoxin).

  1967cited by this paper
• Study of the adrenal non-heme iron protein (adrenodoxin) by electron spin resonance.

  1966cited by this paper
• Enzymatic reduction of non-heme iron protein (adrenodoxin) by reduced nicotinamide adenine dinucleotide phosphate.

  1965cited by this paper
• THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE.

  1964cited by this paper

• Adrenodoxin alters human cytochrome P450 27A1 structure and reaction efficiency beyond supplying electrons.

  2025cites this paper
• Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the P144L mutation of FDX2

  2024cites this paper
• Comparative biochemical characterization of mammalian-derived CYP11A1s with cholesterol side-chain cleavage activities.

  2023cites this paper
• The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive

  2023cites this paper
• Adrenodoxins and Their Role in the Cytochrome P450 Systems

  2022cites this paper
• Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes.

  2020influential citation
• Pregnenolone overproduction in Yarrowia lipolytica by integrative components pairing of cytochrome P450scc system.

  2019cites this paper
• A large-scale comparative analysis of affinity, thermodynamics and functional characteristics of interactions of twelve cytochrome P450 isoforms and their redox partners.

  2019cites this paper
• Human mitochondrial cytochrome P450 27C1 is localized in skin and preferentially desaturates trans-retinol to 3,4-dehydroretinol

  2017cites this paper
• Raman and infrared spectroscopic evidence for the structural changes of the 2Fe2S cluster and its environment during the interaction of adrenodoxin and adrenodoxin reductase.

  2017cites this paper
• A Fluorescent Readout for the Oxidation State of Electron Transporting Proteins in Cell Free Settings.

  2016cites this paper
• Metabolism of Oral Turinabol by Human Steroid Hormone–Synthesizing Cytochrome P450 Enzymes

  2016cites this paper
• Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

  2016cites this paper
• Metabolism of oral-turinabol by human steroid hormone-synthesizing cytochromes P450 •

  2015influential citation
• 2β- and 16β-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation.

  2015cites this paper
• Human steroidogenic cytochromes P450 : biotransformation of drugs and biotechnological application

  2015cites this paper
• Mammalian and Bacterial Cytochromes P450 Involved in Steroid Hydroxylation: Regulation of Catalysis and Selectivity, and Potential Applications

  2014cites this paper
• Kinetic Rate Constant Prediction Supports the Conformational Selection Mechanism of Protein Binding

  2012cites this paper
• Adrenodoxin—A versatile ferredoxin

  2012cites this paper
• uman aldosterone synthase : Recombinant expression in E . coli and purification nables a detailed biochemical analysis of the protein on the molecular level

  2012cites this paper
• Kinetic and optical biosensor study of adrenodoxin mutant AdxS112W displaying an enhanced interaction towards the cholesterol side chain cleavage enzyme (CYP11A1)

  2011influential citation
• Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins.

  2011cites this paper
• Structural biology of redox partner interactions in P450cam monooxygenase: a fresh look at an old system.

  2011cites this paper
• Application of AFM and optical biosensor for investigation of complexes formed in P450-containing monooxygenase systems.

  2011cites this paper
• Interactions of natural polyamines with mammalian proteins

  2011influential citation
• Predicting and characterising protein-protein complexes

  2011cites this paper
• A structure‐based benchmark for protein–protein binding affinity

  2011cites this paper
• Polyamines: naturally occurring small molecule modulators of electrostatic protein-protein interactions.

  2010cites this paper
• A solution model of the complex formed by adrenodoxin and adrenodoxin reductase determined by paramagnetic NMR spectroscopy.

  2010cites this paper
• Atomic Force Microscopy Study of Protein–Protein Interactions in the Cytochrome CYP11A1 (P450scc)-Containing Steroid Hydroxylase System

  2010cites this paper
• Productive and non-productive complexes in cytochrome P450-containing systems

  2009cites this paper
• The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer.

  2009cites this paper
• Electron Transfer between Cytochrome P450cin and Its FMN-containing Redox Partner, Cindoxin*

  2007cites this paper
• Effects of the common pesticide methoxychlor on ovarian steroidogenesis

  2007cites this paper
• Protein phosphorylation and intermolecular electron transfer: a joint experimental and computational study of a hormone biosynthesis pathway.

  2007cites this paper
• Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

  2007influential citation
• Interaction between mitochondrial CYP27B1 and adrenodoxin: role of arginine 458 of mouse CYP27B1.

  2006cites this paper
• Antioxidant Protective Mechanisms against Reactive Oxygen Species (ROS) Generated by Mitochondrial P450 Systems in Steroidogenic Cells

  2006cites this paper
• Cytochromes P450 as versatile biocatalysts.

  2006cites this paper
• Survey of the year 2004 commercial optical biosensor literature

  2005cites this paper
• Survey of the year 2003 commercial optical biosensor literature

  2005cites this paper
• The interaction domain of the redox protein adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor adrenodoxin reductase.

  2005cites this paper