Rubredoxins belong to the simplest class of iron-sulfur proteins. They contain a single iron coordinated by four cysteinate sulfurs. The rubredoxin from Clostridium pasteurianum was overproduced in Escherichia coli, and the metal was incorporated into the apoprotein by in vitro reconstitution. Protein samples were prepared at natural isotopic abundance, 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 hyperfine-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 labeled selectively with deuterium. The cysteine hydrogen signals are resolved more clearly by H-2 (lower magnetogyric ratio) than by H-1 (higher magnetogyric ratio) NMR spectroscopy. In the oxidized state, signals from two of the four ct-hydrogens are located downfield in the 150-200 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-hydrogens 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 were 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 ligate the iron on the basis of selective labeling with [N-15]cysteine.