DETECTION AND CLASSIFICATION OF HYPERFINE-SHIFTED H-1, H-2, AND N-15 RESONANCES FROM THE 4 CYSTEINES THAT LIGATE IRON IN OXIDIZED AND REDUCED CLOSTRIDIUM-PASTEURIANUM RUBREDOXIN

Rubredoxins belong to the simplest class of iron-sulfur proteins. They contain a single iron coordinated by four cysteinate sulfurs. The rub redoxin from Clostridium pasteurianum was overproduced in Escherichia coli, and the metal was incorporated into the apoprotein by in vitro r econstitution. Protein samples were prepared at natural isotopic abund ance, labeled uniformly with N-15, and labeled specifically with [H-2( alpha)]cysteine, [H-2(beta 2,beta 3)]cysteine, and [N-15]cysteine. One -dimensional H-1, H-2, and N-15 nuclear magnetic spectroscopy was used to study the electron-nuclear interactions. Previously unreported hyp erfine-shifted resonance signals were observed in the H-1 and H-2 NMR spectra of rubredoxin samples in both the oxidized and reduced states. Signals from the alpha- and beta-hydrogens of the four cysteines were identified unambiguously from H-1 and H-2 NMR spectra of samples labe led selectively with deuterium. The cysteine hydrogen signals are reso lved more clearly by H-2 (lower magnetogyric ratio) than by H-1 (highe r magnetogyric ratio) NMR spectroscopy. In the oxidized state, signals from two of the four ct-hydrogens are located downfield in the 150-20 0 ppm range; the other two are found upfield at about -10 ppm. Signals from all eight beta-hydrogens were detected downfield in the 300-900 ppm region. Upon reduction, the H-1 NMR signals from all eight beta-hy drogens lie downfield between 150 and 240 ppm; signals from two of the four a-hydrogens lie upfield near 0 ppm, and those from the other two are downfield at 16 and 19 ppm. Thirteen hyperfine-shifted signals we re resolved in one-dimensional N-15 NMR spectra of the sample labeled uniformly with N-15. The two signals located farthest upfield and two signals in the downfield region were assigned to the cysteines that li gate the iron on the basis of selective labeling with [N-15]cysteine.