The rubredoxin from Clostridium pasteurianum (CpRd) provides an excellent system for investigating how the protein sequence modulates the reduction potential of the active site in an iron-sulfur protein. 15 N NMR spectroscopy has allowed us to determine with unprecedented accuracy the strengths of all six key hydrogen bonds between protein backbone amides and the sulfur atoms of the four cysteine residues that ligate the iron in the oxidized (Fe III ) and reduced (Fe II ) forms of wild-type CpRd and nine mutants (V44G, V44A, V44I, V44L, V8G, V8A, V8I, V8L, and V8G͞V44G). The length (or strength) of each hydrogen bond was inferred from the magnitude of electron spin delocalized across the hydrogen bond from the iron atom onto the nitrogen. The aggregate lengths of these six hydrogen bonds are shorter in both oxidation states in variants with higher reduction potential than in those with lower reduction potential. Differences in aggregate hydrogen bonding upon reduction correlate linearly with the published reduction potentials for the 10 CpRd variants, which span 126 mV. Sequence effects on the reduction potential can be explained fully by their influence on hydrogen-bond strengths.iron-sulfur protein ͉ reduction potential tuning ͉ 15 N NMR ͉ paramagnetic NMR

15N NMR hyperfine-shift data were collected for wild-type and site-specific mutant (V44I, V44A, and V44G) Clostridium pasteurianum rubredoxins in the oxidized state. Whereas most of the (15)N NMR signals did not exhibit large systematic changes upon mutation of residue 44, the signal from the backbone nitrogen of residue 44 itself (arrows) shifted by approximately 400 ppm. These shifts were used to determine the lengths of the hydrogen bond between the backbone amide of residue 44 and the side-chain sulfur of cysteine-44, which is covalently ligated to the iron of the metal center. The results, which demonstrated that this hydrogen bond is shorter in mutants with higher reduction potential, point to the importance of hydrogen bonds in modulating the reduction potential of iron-sulfur proteins.

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.

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