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Of the aromatic 'H-NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a throughspace ring-current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P-450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation.As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P-45OlI, (CYPllB1)-dependent and P-450,,, (CYP1 lA1)-dependent substrate conversions were studied. No influence on V,,, values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the K, values were increased, as was the K, value for binding of CYPl 1Al to the IH56Tladreno-doxin.The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary-structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent-exposed region of the molecule.Keyword,s. Adrenodoxin ; 'H-NMR ; aromatic region ; fluorescence.Adrenodoxin is a member of the ferredoxin family of proteins, that are widely distributed in bacteria, plants and animals. As a rule, the ferredoxins are low-molecular-mass proteins (6000-25 000 Da) that are negatively charged at neutral pH and all contain iron-sulfur clusters as the redox-active group. Bovine adrenodoxin is involved in two electron-transfer systems in the inner mitochondrial membrane of bovine adrenal cortex. Both systems contain NADPH-dependent adrenodoxin reductase, adrenodoxin and the specific cytochrome P-450, cytochrome P-450,,, (CYPllAl) or cytochrome P-45OI,,, (CYPllBl).The three-dimensional structure of adrenodoxin has not yet been elucidated and there are few data on the structural basis of the mechanism of protein-protein interaction among adrenodoxin and its redox partners. The shuttle model (Lambeth et al., 1979;Hanukoglu and Jefcoate, 1980), a ternary complex formation of adrenodoxin reductase, adrenodoxin, and the cytochrome P-450 (Kido and Kimura, 1979), and a model suggesting Correspondence to R. Bemhardt, Max-Delbriick-Centrum fur Molekulare Medizin,

An analysis of the electron transfer kinetics from the reduced [2Fe-2S] center of bovine adrenodoxin and its mutants to the natural electron acceptors, cytochromes P450scc and P45011␤, is the primary focus of this paper. A series of mutant proteins with distinctive structural parameters such as redox potential, microenvironment of the iron-sulfur cluster, electrostatic properties, and conformational stability was used to provide more detailed insight into the contribution of the electronic and conformational states of adrenodoxin to the driving forces of the complex formation of reduced adrenodoxin with cytochromes P450scc and P45011␤ and electron transfer. The apparent rate constants of P450scc reduction were generally proportional to the adrenodoxin redox potential under conditions in which the proteinprotein interactions were not affected. However, the effect of redox potential differences was shown to be masked by structural and electrostatic effects. In contrast, no correlation of the reduction rates of P45011␤ with the redox potential of adrenodoxin mutants was found. Compared with the interaction with P450scc, however, the hydrophobic protein region between the iron-sulfur cluster and the acidic site on the surface of adrenodoxin seems to play an important role for precise complementarity in the tightly associated complex with P45011␤.The key step of different biological reactions such as oxidative phosphorylation, photosynthesis, respiration, drug metabolism, and many other processes taking place in living organisms is an electron transfer. Electron transfer reactions commonly occur between protein-bound prosthetic groups of the electron donors and acceptors by protein-protein interaction (1). However, only a little is known about the geometry of the protein-protein complex required for successful electron transfer and about the rate-limiting processes.Bovine adrenodoxin is of particular interest because of its specific role as an electron carrier associated with steroid hydroxylation reactions. Adrenodoxin is a member of the ferredoxin family of electron-transferring non-heme iron proteins. It is a low molecular mass protein (14 kDa), negatively charged at neutral pH values and containing the [2Fe-2S] cluster as redoxactive group. Adrenodoxin mediates the transfer of electrons from NADPH via adrenodoxin reductase to the heme iron of mitochondrial cytochromes P450, which are localized in the inner mitochondrial membrane of bovine adrenal cortex, involved in the side chain cleavage of cholesterol (CYP11A1), 1 the 11␤-hydroxylation of 11-deoxycorticosterone and 11-deoxycortisol, and the production of aldosterone (CYP11B1). The flavin prosthetic group of adrenodoxin reductase accepts two electrons from NADPH and contributes one electron to adrenodoxin. Reduced adrenodoxin in turn transfers an electron to either ferric or oxygen-bound P450 species. Both side chain cleavage and 11␤-hydroxylation utilize molecular oxygen and require two electrons per oxidation (six electrons in total for side chain cleavage) (...

Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-1 14 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 "C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change AC,, = 7.28 2 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (AC, = 4.29 2 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.Keywords: ferredoxin; iron-sulfur protein; mutants; protein unfolding; scanning microcalorimetryThe importance of side-chain hydrogen bonds for protein stability was considered by Perutz and Raid (1975) by example of bacterial ferredoxins. Unfortunately, attempts to study ferredoxins by means of thermodynamic approaches failed due to destruction of the ironsulfur cluster prior to protein unfolding. Only recently was an approach developed by example of adrenal ferredoxin that enables unfolding studies by means of high-sensitivity scanning microcalorimetry (Burova et al., 1995). Independently, Anabaena ferredoxin was studied by denaturant-induced unfolding (Hurley et al., 1995).Adrenal ferredoxin (Adx) is a small acidic protein consisting of 128 amino acid residues and an iron-sulfur cluster of the [2Fe-2S] type (Okamura et al., 1985). The protein participates in the synthesis of steroid hormones by mediating the electron transport from the NADPH-dependent adrenodoxin reductase to mitochondrial cytochromes P450 (Estabrook et al., 1973;Lambeth, 1990;Usanov et al., 1990). Accordingly, most investigations on Adx Reprint requests to: Wolfgang Pfeil, Max-Delbriick-Centre for Molecular Medicine, Robert-Rossle-Street 10, D-13125 Berlin-Buch, Germany; e-mail: wpfeil@orion.rz.mdc-berlin.de.Abbreviations: Adx, adrenal ferredoxin (adrenodoxin); DSC, differential scanning microcalorimetry; EPR, electron paramagnetic resonance; Gu-HCI, guanidine hydrochloride; K,, Michaelis-Menten constant; V,,, Michaelis-Menten constant; T,,, transition temperature ("C); AE, redox potential (mv); AH, denaturation enthalpy, calorimetric value (kJ/mol); Ah, specific denaturation enthalpy (J/g); CR, cooperative ratio; AC,,, heat capacity change (kJ/mol/K); C, , , , molar excess heat capacity; u, standard deviation, percent of C,,,,,.were devoted to electron transfer and interactions within the system consisting of adrenodoxin reductase, Adx, and cytochrome P450 (Estabrook et al., 1973;Kid0 & Kimura, 1979;Hanukoglu & Jefcoate, 1980;Lambeth & Pember, 1983;Lambeth & Kriengsiri, 1985;Usanov et al., 1986;Hara & Kimura, 1989). Because wildtype Adx was expressed in Escher...