A Conserved Histidine in Vertebrate-Type Ferredoxins Is Critical for Redox-Dependent Dynamics ² Milka Kostic, ‡ Rita Bernhardt, § and Thomas C. Pochapsky* ,‡ Department of Chemistry, Brandeis UniVersity, 415 South Street, MS 015, Waltham, Massachusetts 02451, and FR 8.8 Biochemie, UniVersita ¨t des Saarlandes, Postfach 151150, D-66041, Saarbru ¨cken, Germany ReceiVed March 28, 2003; ReVised Manuscript ReceiVed May 16, 2003 ABSTRACT: Adrenodoxin (Adx) belongs to the family of Cys 4 Fe 2 S 2 vertebrate-type ferredoxins that shuttle electrons from NAD(P)H-dependent reductases to cytochrome P450 enzymes. The vertebrate-type ferredoxins contain a conserved basic residue, usually a histidine, adjacent to the third cysteine ligand of the Cys 4 Fe 2 S 2 cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogen- bonding network within the domain of Adx that interacts with redox partners. It has been proposed that this network acts as a mechanical link between the metal cluster binding site and the interaction domain, transmitting redox-dependent conformational or dynamical changes from the cluster binding loop to the interaction domain. H/D exchange studies indicate that oxidized Adx (Adx o ) is more dynamic than reduced Adx (Adx r ) on the kilosecond time scale in many regions of the protein, including the interaction domain. Dynamical differences on picosecond to nanosecond time scales between the oxidized (Adx o ) and reduced (Adx r ) adrenodoxin were probed by measurement of 15 N relaxation parameters. Significant differences between 15 N R 2 rates were observed for all residues that could be measured, with those rates being faster in Adx o than in Adx r . Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between 15 N R 2 rates or H/D exchange rates were observed in either mutant, indicating that His 56 is required for the redox-dependent behavior observed in WT Adx. Comparison of chemical shift differences between oxidized and reduced H56Q and H56R Adx confirms that redox- dependent changes are smaller in these mutants than in the wild-type Adx. The oxidative conversion of cholesterol to pregnenolone in mitochondria of the adrenal cortex marks the beginning of steroid hormone biosynthesis. Cytochrome P-450 scc (CYP11A1) catalyzes this three-step oxidation. Cholesterol is first converted to 22(R)-hydroxycholesterol, which is further oxidized to 20,22-dihydroxycholesterol and finally to pregnenolone and 4-methylpentanal. Each oxidation requires a molecule of O 2 and two electrons. The electrons required are ultimately derived from the oxidation of NADPH, which is catalyzed by the flavoenzyme, adreno- doxin reductase (AdR). 1 AdR reduces the Cys 4 Fe 2 S 2 ferre- doxin Adx, which acts as a single-electron shuttle from AdR to CYP11A1. Besides CYP11A1, bovine Adx is the reductant of another cytochrome P-450 enzyme, P-450 11 (CYP11B1). This enzyme catalyzes the 11- and C-18 hydroxylation of steroids, leading to formation of cortisol and aldosterone. All three components of the electron-transfer chain are located on the matrix side of the inner mitochondrial membrane, mostly in the adrenal cortex, although they can be found in the mitochondria of some other tissues as well (1). The cognate P450s of the soluble Adx are membrane- bound enzymes, and adrenodoxin reductase (AdR) is mem- brane associated. There are several proposed models for electron transport in this system. The most widely accepted is the shuttle model, wherein Adx acts as a mobile shuttle between AdR and cytochrome P450 (2, 3). Recently, it was observed that Adx forms dimers when oxidized, which lead to another model for electron transfer (4) in which oxidized Adx (Adx o ) binds to the AdR as a dimer. A single electron is transferred from AdR to the directly bound Adx and then to the second Adx molecule. Upon reduction, reduced Adx (Adx r ) dissociates, and the second electron is transferred from AdR to Adx, which dissociates from AdR upon reduction. Both Adx r monomers transfer one electron each to the cytochrome P450 and then redimerize. It has been pointed out that the mechanism of electron transfer might differ from one mitochondrial P450 system to another (5). Adx is the archetype of the family of vertebrate-type ferredoxins. Mature bovine Adx consists of 128 amino acid residues and is negatively charged at physiological pH. It contains a single Fe 2 S 2 cluster ligated by four cysteinyl thiolate ligands, Cys 46, Cys 52, Cys 55, and Cys 92. The metal cluster cycles between two oxidation states, Fe(III)/ Fe(III) and Fe(II)/Fe(III). Crystal structures of a C-terminal ² This work was supported by a grant from the National Institutes of Health (RO1-GM44191 to T.C.P.) and by a grant from the Deutsche Forschungsgemeinschaft (Be 1343/12-1 to R.B.). * To whom correspondence should be addressed. Phone: (781) 736- 2559. Fax: (781) 726-2516. E-mail: pochapsk@brandeis.edu. ‡ Brandeis University. § Universita ¨t des Saarlandes. 1 Abbreviations: Adx, adrenodoxin; DTT, dithiothreitol; HSQC, heteronuclear single-quantum coherence; IPTG, isopropyl -D-thioga- lactoside; LB, Luria-Bertani media; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; WT, wild type; Adx o , oxidized adreno- doxin; Adx r , reduced adrenodoxin; RMSD, root mean square deviation; 2D, two dimensional; Pdx, putidaredoxin; Fdx, ferredoxin; AER, atomic element radius; AdR, adrenodoxin reductase; OD, optical density. 8171 Biochemistry 2003, 42, 8171-8182 10.1021/bi034500r CCC: $25.00 © 2003 American Chemical Society Published on Web 06/19/2003