The Tertiary Structure of Full-Length Bovine Adrenodoxin Suggests Functional Dimers 1,2 Irina A. Pikuleva, 3 Kris Tesh, 4 Michael R. Waterman, and Youngchang Kim Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received August 25, 1999, and in revised form September 28, 1999 The three-dimensional X-ray crystal structure of full-length oxidized bovine adrenodoxin (Adx) has been determined at 2.5 Å resolution by molecular re- placement using a structure of a truncated form as a starting model. Crystals of Adx belong to a primitive monoclinic space group P2 1 with four Adx molecules in an asymmetric unit. The unit cell dimensions are a  59.44 Å, b  77.03 Å, c  59.68 Å, and   94.83°. The structure has been refined to an R factor of 23.5%. Structures of the four molecules of full-length Adx (127 amino acids) in the asymmetric unit were compared with each other and also with that of the truncated Adx (4 –108). The overall topology of full-length Adx remains the same as described earlier for the trun- cated protein. Differences that do occur are almost wholly confined to alternate side-chain conformations that reflect differing lattice contacts made by two pro- teins. Extensive interactions found between molecules 1 and 2 in the full-length Adx asymmetric unit may reflect the ability of Adx to form dimers in vivo and are consistent with hydrodynamic measurements which show that in solution there is an equilibrium between monomeric and dimeric forms of Adx. Dimerization of Adx could explain why the truncated form has greater affinity for the P450 redox partner than the full-length form. From these results it can be considered that the mechanism of electron transfer is not necessarily the same in different mitochondrial P450 systems. © 2000 Academic Press Key Words: adrenodoxin; tertiary structure; electron transfer. Ferredoxins are ubiquitous proteins which partici- pate as electron carriers in biological electron transfer reactions in bacteria, plants, and animals. They are classified by the number and types of iron–sulfur clus- ters they contain. Mammalian [2FeO2S] ferredoxins are small 13- to 14-kDa soluble proteins present in the mitochondrial matrix where they transfer electrons from ferredoxin reductase to mitochondrial forms of cytochrome P450 (P450). Mitochondrial P450s play im- portant physiological roles, being involved in the bio- synthesis of steroid hormones, the production of bile acids, and the formation of vitamin D 3 metabolites. Whereas different forms of P450 are necessary to me- tabolize different substrates, a single form of ferre- doxin functions as the mitochondrial P450 redox part- ner. There is a high degree of sequence identity (90%) between mammalian ferredoxins from different species (1). Human and bovine ferredoxins have very similar properties and can substitute for each other in electron transfer reactions in reconstituted systems with ferre- doxin reductase and P450 (2). The ferredoxin from bovine adrenal cortex, desig- nated adrenodoxin (Adx), 5 is the most extensively char- acterized mammalian ferredoxin because of its relative 1 This study was supported by NIH Grants GM37942 and ES00267. 2 The X-ray coordinates have been deposited in the Brookhaven Protein Data Bank (PDB ID Code 1cje). 3 To whom correspondence should be addressed at permanent ad- dress: Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555- 1031. Fax: (409) 772-9642. E-mail: irpikule@utmb.edu. 4 Permanent address: Molecular Structure Corp., 3200 Research Forest Dr., The Woodlands, TX 77381. 5 Abbreviations used: Adx, adrenodoxin; Adr, adrenodoxin reduc- tase; P450scc, cholesterol side-chain cleavage cytochrome P450, product of the CYP11A1 gene; P450c11, cytochrome P450 catalyzing the 11-hydroxylation of 11-deoxycortisol to cortisol, product of the CYP11B1 gene; P450aldo, cytochrome P450 catalyzing the produc- tion of aldosterone, product of the CYP11B2 gene; P450c27, cyto- chrome P450 catalyzing the multiple oxidation reactions at C-27 of steroids, product of the CYP27A1 gene (48); MALDI-TOF–MS, ma- trix-assisted laser desorption ionization time-of-flight–mass spec- trometry; rms, root mean square; KPB, potassium phosphate buffer, pH 7.4; MAD, multiple-wavelength anomalous dispersion. 44 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 373, No. 1, January 1, pp. 44 –55, 2000 Article ID abbi.1999.1536, available online at http://www.idealibrary.com on