The four cysteine ligands which coordinate the Fe atom in the electron-transfer protein rubredoxin lie on loops of the polypeptide which form approximate local twofold symmetry. The cysteine ligands in the protein from Clostridium pasteurianum lie at positions 6, 9, 39 and 42. Two glycine residues adjacent to the cysteine ligands at positions 10 and 43 are conserved in all rubredoxins, consistent with the proposal that a beta-carbon substituent at these positions would eclipse adjacent peptide carbonyl groups [Adman et al. (1975). Proc. Natl Acad. Sci. USA, 72, 4854-4858]. X-ray crystal structures of the three mutant proteins G10A, G43A and G10VG43A are reported. The crystal structures of the single-site mutations are isomorphous with the native protein, space group R3; unit-cell parameters are a = 64.3, c = 32.9 A for G10A and a = 64.4, c = 32.8 A for G43A. The crystals of the double mutant, G10VG43A, were in space group P43212, unit-cell parameters a = 61.9, c = 80.5 A, with two molecules per asymmetric unit. The observed structural perturbations support the hypothesis that mutation of the conserved glycine residues would introduce strain into the polypeptide. In particular, in the G10VG43A protein substitution of valine at Gly10 causes the 9-10 peptide link to invert, relieving steric interaction between Cys9 O and Val10 Cbeta. This dramatic change in conformation is accompanied by the loss of the 10N-HcO6 hydrogen bond, part of the chelate loop Thr5-Tyr11. The new conformation allows retention of the 11N-HcS9 hydrogen bond, but converts it from a type II to a type I hydrogen bond. This occurs at the cost of a less tightly packed structure. The structural insights allow rationalization of 1H NMR data reported previously for the 113CdII-substituted proteins and of the negative shifts observed in the FeIII/FeII mid-point potentials upon mutation.