The Rubredoxin from <i>Clostridium pasteurianum</i>:  Mutation of the Iron Cysteinyl Ligands to Serine. Crystal and Molecular Structures of Oxidized and Dithionite-Treated Forms of the Cys42Ser Mutant

Abstract

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 Fe III (S γ -Cys) 3 (O γ -Ser) center (Fe−O, 1.82(8) Å). Significant structural change is restricted to the region around the mutation. EXAFS experiments confirm Fe III S 3 O (O = O γ -Ser or OH x ) centers in each oxidized protein at pH 8. The reduction potentials of the Fe III/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 p K a red values of about 9 and 7. EXAFS data indicate an increase of 0.2−0.3 Å in the Fe II −O distances in passing through these characteristic p K a red values. 1 H NMR experiments on Cd II forms reveal the presence of Cd II (S-Cys) 3 {O(H)-Ser} centers in the surface ligand mutants C9S and C42S by the detection of 113 Cd−O−CH β 2 coupling and S−OH γ resonances. The assumption of the presence of Fe II (S-Cys) 3 (O-Ser) centers in each mutant protein at pH values above the characteristic p K a red allows a simple interpretation of the electrochemical behavior. Protonation of the Fe−O γ -Ser link upon reduction is proposed, followed by hydrolysis at lower pH values: Fe III −O γ -Ser + H + + e - → Fe II −O γ (H)-Ser; Fe II −O γ (H)-Ser + H 2 O → Fe II −OH 2 + HO γ -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 Fe II -C42S protein by treatment of Fe III -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 SO 2, a result confirmed by reactions in solution: Cys-SH + SO 2 → Cys-S II −S IV O 2 - + H + .