Isolation is reported of the four mutant proteins of the electron-transfer protein rubredoxin from Clostridium pasteurianum in which each of the four cysteine ligands is changed in turn to serine. They fall into two pairs whose properties depend on whether an interior (C6, C39) or a surface (C9, C42) cysteine ligand is substituted. A crystal structure of the oxidized C42S protein (1.65 Å; R, 18.5%) confirms the presence of an FeIII(Sγ-Cys)3(Oγ-Ser) center (Fe−O, 1.82(8) Å). Significant structural change is restricted to the region around the mutation. EXAFS experiments confirm FeIIIS3O (O = Oγ-Ser or OH x ) centers in each oxidized protein at pH 8. The reduction potentials of the FeIII/II couple are decreased by about 100 and 200 mV, respectively, in the interior and surface ligand mutants. The potentials are pH-dependent with respective pK a red values of about 9 and 7. EXAFS data indicate an increase of 0.2−0.3 Å in the FeII−O distances in passing through these characteristic pK a red values. 1H NMR experiments on CdII forms reveal the presence of CdII(S-Cys)3{O(H)-Ser} centers in the surface ligand mutants C9S and C42S by the detection of 113Cd−O−CHβ 2 coupling and S−OHγ resonances. The assumption of the presence of FeII(S-Cys)3(O-Ser) centers in each mutant protein at pH values above the characteristic pK a red allows a simple interpretation of the electrochemical behavior. Protonation of the Fe−Oγ-Ser link upon reduction is proposed, followed by hydrolysis at lower pH values:  FeIII−Oγ-Ser + H+ + e- → FeII−Oγ(H)-Ser; FeII−Oγ(H)-Ser + H2O → FeII−OH2 + HOγ-Ser. The differences in reduction potentials, their pH dependence, and the onset of irreversible electrochemistry can be attributed to differences in the Fe−O bonds of the interior and surface ligands. These differences appear to result from variation in the conformational flexibility of the protein chelate loops which carry the ligands. An attempt to generate crystals of the reduced FeII-C42S protein by treatment of FeIII-C42S crystals with dithionite at pH 4 led to loss of iron. A crystal structure (1.6 Å; R, 16.8%) reveals that cysteine residues 6 and 9 have trapped the oxidation product SO2, a result confirmed by reactions in solution:  Cys-SH + SO2 → Cys-SII−SIVO2 - + H+.

Understanding the structure and dynamics of the enzymes that mediate antibiotic resistance of pathogenic bacteria will allow us to take steps to combat this increasingly serious public health hazard. Complete backbone NMR resonance assignments have been made for the broad-specificity metallo-beta-lactamase CcrA from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. Chemical shift indices show that the secondary structure of the CcrA molecule in solution is very similar to that in published crystal structures. A loop adjacent to the two-zinc catalytic site exhibits significant structural variation in the published structures, but appears from the NMR experiments to be a regular beta-hairpin. Backbone heteronuclear NOE measurements indicate that this region has slightly greater flexibility on a picosecond to nanosecond time scale than the molecule as a whole. The loop appears to have an important role in the binding of substrates and inhibitors. Binding of the inhibitor 3-[2'-(S)-benzyl-3'-mercaptopropanoyl]-4-(S)-carboxy-5, 5-dimethylthiazolidine causes a marked increase in the stability of the protein toward unfolding and aggregation, and causes changes in the NMR resonance frequencies of residues close to the active (zinc-binding) site, including the beta-hairpin loop. There is a small but significant increase in the heteronuclear NOE for this loop upon inhibitor binding, indicative of a decrease in flexibility. In particular, the NOE of the indole ring of tryptophan 49, at the tip of the beta-hairpin loop, changes from a low value characteristic of a random coil chain to a significantly higher value, close to that observed for the backbone amides in this region of the protein. These results strongly suggest that the hairpin loop participates in the binding of substrate and in the shielding of the zinc sites from solvent. The broad specificity of the CcrA metallo-beta-lactamase may in fact reside in the plasticity of this part of the protein, which allows it to accommodate and bind tightly to substrates of a variety of shapes and sizes.

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 βand γ-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. 1 H 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 f V mutation has a larger effect than a G f 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.