Bovine, porcine and sheep adrenodoxin, and the trypsin-resistant form of bovine adrenodoxin have been studied by one-and two-dimensional 'H-NMR spectroscopy. Assignment of the resonances for all the aromatic amino acids with resolved aromatic resonances have been made by correlating NMR spectra with the amino acid sequences from various species. Slowly exchanging amide protons and downfield shifted a-protons of His10 and Phell suggest possible involvement in /I-sheet structure. The effects on the assigned resonances due to the specific spin-label with a nitroxide radical at Cys95 have been analyzed on a two-dimensional 'H-NMR spectrum. The present results provide evidence for a structural similarity with a model for the structure of adrenodoxin based on a sequence alignment with that of Spirulina platensis ferredoxin, for which X-ray crystallographic data is available. &-Methyl groups of Met120 and Met122 have been assigned by comparing 'H-NMR spectra of adrenodoxin with those of the trypsin-resistant form of adrenodoxin which is specifically cleaved at Argll5. EMethyl groups of Met120 and Met122 have an exceptionally long longitudinal relaxation time compared with those of valyl and leucyl methyl groups, suggesting that the COOH-terminal peptide spanning over 13 amino acids rotates rather freely in the solvent.Adrenal ferredoxin (adrenodoxin) is a small iron-sulfur protein with relative molecular mass of about 14000, which is located in the matrix of the adrenal cortex mitochondria. Adrenodoxin is synthesized with a signal peptide yielding a precursor protein [I, 21. The mature form, after being processed and translocated into mitochondria, comprises 128 amino acids based on cDNA [3] and amino acid sequencing [4, 51. It functions as an electron transfer protein from NADPHadrenodoxin reductase to cytochromes P-45OsCc (cholesterol side-chain cleavage enzyme) and P-4501 1 B (steroid 11B monooxygenase) in the steroidogenic electron transfer system [6] (and references therein).Similar iron-sulfur proteins with a 2Fe-2S cluster are distributed in a wide range of biological electron-transfer chains including plant and blue green algae [7]. Meyer et al. [8] have divided them into three classes based on the amino acid sequence similarity. Adrenodoxin and putidaredoxin from Pseudomonas putida [9], both participating in hydroxylation reactions, belong to one class. The plant ferredoxins, exemplified by ferredoxins from Spinacea oleracea (spinach) and Correspondence to S. Miura, Kagawa Medical School, Department of Biochemistry, Miki-cho, Kita-gun, Kagawa 761-07, JapanAbbreviations. COSY, J-correlated spectroscopy; DQF, double quantum filtered; NOESY, nuclear Overhauser effect spectrosocopy; HOHAHA, homonuclear Hartmann-Hahn; DANTE, delays alternating with nutations for tailored excitation; CPMG, Carr-PurcellMeilboom-Gill; 4-maleimide TEMP, 4-maleimide-2,2,6,6-tetramethyl-1 -piperidinyloxy.Enzymes. NADPH -adrenodoxin reductase (EC 1.1 8.1.2); cytochrome P-45OsCc cholesterol side-chain cleavage enzyme (EC 1.14.15.6); c...
