Conserved glycine residues at positions 10 and 43 in the electron transfer protein rubredoxin (active site : Fe(Cys-S)(4)) from Clostridium pasteurianum are related by a pseudo-2-fold symmetry. They have been mutated to alanine and valine and four single and two double mutant (G10V/G43A and G10V/G43V) proteins expressed in stable form in Escherichia coli. Physical properties were modified by steric interactions between the beta- and gamma-carbon substituents of the new side chains and the CO functions of C9 and C42 and other adjacent groups. These interactions perturb the chelate loops formed by residues 5-11 and 38-44. H-1 NMR results for Cd(II) forms indicate that the Pr-i side chain of V10 in the G10V mutant occupies the surface pocket defined by loop 5-11 and thereby modifies the environment of the 5-11 NH protons. The equivalent side chain of V43 in G43V is denied the same access to the 38-44 pocket. This leads to a specific perturbation of the V44-NH ... S-C42 interaction in this mutant. These effects are additive in the double mutant G10V/G43V, consistent with the different structural changes being localized in each loop. The midpoint potentials of the iron forms of the six mutants are shifted negatively relative to the recombinant protein by -16 to -86 mV. A G --> V mutation has a larger effect than a G --> A, but again, an additivity of the differential effects is seen in the double mutants. Minor perturbations of resonance Raman and electronic spectra are dominated by the mutation at G10. Overall, the present work represents one approach to the systematic exploration of the influence of the protein chain upon the fundamental properties of this molecule.