No abstract

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,

Site-directed mutagenesis of a domain (amino acids 299-338) aligning to the I-helix region of P450cam, P450BM3 and P450terp was used to investigate the different regioselectivities displayed in the hydroxylation reactions performed by human aldosterone synthase (P450aldo) and 11beta-hydroxylase (P45011beta). The two enzymes are 93% identical and are essential for the synthesis of mineralocorticoids and glucocorticoids in the human adrenal gland. Single replacement of P450aldo residues for P45011 beta-specific residues at positions 296, 301, 302, 320, and 335 only gave rise to slightly increased 11beta-hydroxylase activities. However, a L301P/A320V double substitution increased 11beta-hydroxylase activity to 60% as compared with that of P45011 beta. Additionally substituting Ala-320 for Val-320 of P45011 beta further enhanced this activity to 85%. The aldosterone synthase activities of the mutant P450aldo proteins were suppressed to a varying degree, with triple replacement mutant L301P/E302D/A320V retaining only 10% and double replacement mutant L301P/A320V retaining only 13% of the P450aldo wild type activity. These results demonstrate a switch in regio- and stereoselectivities of the engineered P450aldo enzyme due to manipulation of residues at three critical positions, and we attribute the determination of these features in P450aldo to the structure of a region analogous to the I-helix in P450cam.

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,

Low‐resolution three‐parameter models of the shape of a biopolymer in solution can be determined by a new indirect method from small‐angle X‐ray scattering without contrast‐variation experiments. The basic low‐resolution model employed is a triaxial ellipsoid – the inertia‐equivalent ellipsoid (IEE). The IEE is related to the tensor of inertia of a body and the eigenvalues and eigenvectors of this tensor can be calculated directly from the atomic coordinates and from the homogeneous solvent‐excluded body of a molecule. The IEE defines a mean molecular surface (like the sea level on earth) which models the molecular shape adequately if the IEE volume is not more than 30% larger than the dry volume of the molecule. Approximately 10 to 15% of the solvent‐excluded volume is outside the ellipsoid; the radii of gyration of the IEE and of the homogeneous molecular body are identical. The largest diameter of the IEE is about 5 to 15% (~0.2–0.8 nm) smaller than the maximum dimension of globular molecules with molecular masses smaller than 65000 daltons. From the scattering curve of a molecule in solution the IEE can be determined by a calibration procedure. 29 proteins of known crystal structure have been used as a random sample. Systematic differences between the axes of the IEE, calculated directly from the structure, and the axes of the scattering‐equivalent ellipsoids of revolution, estimated from the scattering curve of the molecule in solution, are used to derive correction factors for the axial dimensions. Distortions of model dimensions of 20 to 40% (up to 1 nm), caused by misinterpretation of scattering contributions from electron density fluctuations within the molecule, are reduced to a quarter by applying these correction factors to the axes of the scattering‐equivalent ellipsoids of revolution. In a computer experiment the axes of the inertia‐equivalent ellipsoids have been determined for a further nine proteins with the same accuracy. The automated estimation of the IEE from the scattering curve of a molecule in solution is realized by the Fortran77 program AUTOIEE.