Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin

Adamovich, T.B.; Pikuleva, Irina A.; Chashchin, Vadim L.; Usanov, Sergey A.

doi:10.1016/0167-4838(89)90254-9

Cited by 34 publications

(28 citation statements)

References 13 publications

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“…The hydrogen bonding interactions predominate upon the Adx-CYP11A1 complex formation. The interface comprises two salt bridges, Lys339 CYP11A1 -Asp72 Adx and Lys343 CYP11A1 -Asp76 Adx; and is consistent with site-directed mutagenesis and chemical modification data (26)(27)(28)(29). Residue Asp79 of Adx interaction domain, which affects Adx-CYP11A1 complex formation and activity (28)(29)(30), is not involved in direct contact but close (approximately 5 Å) to the "meander" region of CYP11A1, more specifically, to Lys406, the latter being implicated in redox partner binding (31) (Fig.…”

Section: Resultssupporting

confidence: 59%

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Strushkevich

Mackenzie

Cherkesova

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

290

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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 Å 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.

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Section: Resultssupporting

confidence: 59%

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Strushkevich

Mackenzie

Cherkesova

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

290

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“…lion of' cyt~hrome b; and =drenodoxln for bindinil with cytochrom¢ P-450scc suilaests that a similar cationic site on the surface of cytochrome P.4~0scc is involved in the alas©clarion with both prot=ins. Indeed, recently we halve shown by chemical modification and cross.linking experiments that positively charged residues of the N-and C.terminal sequence of cytochrome P.450scc are involved in the electrostatic interaction with adrenodoxin [8,9], The similancy of the processes of the interaction of cytochrome P.4505cc with cytochrom¢ b= and adrenodoxin allowed us to propose that adrenodoxin m~$ht interact with cyLochrome P.450 from endopiasmic reticulum membranes. Indeed, the mteraclion of adrenodoxin with some microsomal cytochrome P-450 isozymes has been demonstrated using spectral titration, affinity, chromatography and reconst~tution experiments (unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Interaction of cytochrome P‐450scc with cytochrome b_s

Усанов¹,

Chashchin²

1991

FEBS Letters

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Received 26 No~rnb~r I~)0 ,~r.~trophcstometrl¢, ~lttntt)r ~,hromalOllr~phy and eros~,hnklnil exl~rm~nls provided e~M,~nu Ih~t cyh~hton~ P,4~0w¢ fron'~ b~ln¢ ~dren~ort). ¢al mtt~htmdrta farms a Iqlhl eumple~c with =yt~hrame ~ from r~bblt h~er rnlcrmomes l)~ the r~or, s0tuled system fholest=r¢~l aside chain acthrlly ol'c)'tuchrome P-4SOscc was ~nhanc~l b~' the atldlllOn of~yt~hrorne b, Cyt~hrome P.4~IO,~, Cytoehf~me b,, t.:~mplex formation, Arltnhy ~hromatollraph),, Chemical woss.hnklnll I, INTRODUCTIONCytochrom¢ P.4S0.dependent monooxygenases can be classified into two large groups [I}'. 0) 'ferredoxindependent' monooxygenases each consistmg of a t'lavoprotein, an iron.sulfur protein, and cytoehrome P.4S0; (h) 'm=crosomaP monooxygenases each containin8 only a flavoprotein and cytochrome P-450. Interrelation between these two groups of monooxygenases, regulat=on of the electron transfer in the membranes of endoplasmic reticulum and mitochondria, and interaction of the two groups of monooxygenases are unclear. Here we report that cytochrome P-4$0ssc from bovine adrenocort=cal mttochondria can form a tight complex wtth cytochrome bs from rabbtt hver mtcrosomes. Our results are consistent with the hypothesis that there =s an interactton between 'microsomal' and 'ferredoxmdependent' monooxygenases and that cytochrome b~ may function as a mobtle earner between microsomes and mttochondrta. MATERIALS AND METHODS 1 C/)emtca/$Thyopropyl-Sepharose, Scpharose 4B, CNBr activated Sepharose 4B, Sephadex G-25 (free) were f-orn Pharmacla Sodmrn cholate, cholesterol, pregnenolone, Tween 20, EDC from Serva P.rfftcatJon of mtcrosoma/and mttochondrlal enzymesCytochrome P-450scc was purified from bovine adrenocortlcal mttochondrta to electrophoretic homogenetty on immobilized adrenodoxm and cholate-Sepharose as described previously [2]. Intact cytochrome bs (d-bat containing the hydrophoblc C-terminal sequence was purified as a by-product of punflcatmn of cytochrome 220045, USSRAbbrevtatton" EDC, l.ethyl-3-(dlrnethylarnmopropyl)carbod.rntde 2,3 Immobih~tlotl of ¢ytochrom¢ P.4JO$ce add eytochrome bjCytoclname P.4S0~:e tva= Immobihzed o. CNBr.acttv=ted $

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“…This observation is consistent with the ionic nature of the complex between Adx and CYP11A1. [64][65][66][67][68] …”

Section: Functional Characterization Of the Interaction Between Adx Amentioning

confidence: 99%

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

et al. 2007

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Protein phosphorylation is a common regulator of enzyme activity. Chemical modification of a protein surface, including phosphorylation, could alter the function of biological electron-transfer reactions. However, the sensitivity of intermolecular electron-transfer kinetics to post-translational protein modifications has not been widely investigated. We have therefore combined experimental and computational studies to assess the potential role of phosphorylation in electron-transfer reactions. We investigated the steroid hydroxylating system from bovine adrenal glands, which consists of adrenodoxin (Adx), adrenodoxin reductase (AdR), and a cytochrome P450, CYP11A1. We focused on the phosphorylation of Adx at Thr-71, since this residue is located in the acidic interaction domain of Adx, and a recent study has demonstrated that this residue is phosphorylated by casein kinase 2 (CK2) in vitro. 1 Optical biosensor experiments indicate that the presence of this phosphorylation slightly increases the binding affinity of oxidized Adx with CYP11A1 ox but not AdR ox . This tendency was confirmed by K A values extracted from Adx concentration-dependent stopped-flow experiments that characterize the interaction between AdR red and Adx ox or between Adx red and CYP11A1 ox . In addition, acceleration of the electron-transfer kinetics measured with stopped-flow is seen only for the phosphorylated Adx-CYP11A1 reaction. Biphasic reaction kinetics are observed only when Adx is phosphorylated at Thr-71, and the Brownian dynamics (BD) simulations suggest that this phosphorylation may enhance the formation of a secondary Adx-CYP11A1 binding complex that provides an additional electron-transfer pathway with enhanced coupling.

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Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin

Cited by 34 publications

References 13 publications

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Interaction of cytochrome P‐450scc with cytochrome b_s

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

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Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin

Cited by 34 publications

References 13 publications

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

Interaction of cytochrome P‐450scc with cytochrome bs

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

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Interaction of cytochrome P‐450scc with cytochrome b_s