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+.

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Redox reaction volumes, obtained by high-pressure cyclic voltammetry, are reported for a selection tris(diimine), tris(diamine), hexaammine, and hexaaqua couples of Fe(III/II), Cr(III/II), Ru(III/II), and Co(III/II). Separation of the intrinsic and electrostrictive volume contributions for these couples has been achieved, some in both aqueous and acetonitrile solutions. For the Co(phen)(3)(3+/2+) system, the intrinsic volume change is estimated to be +15.3 +/- 2.1 cm(3) mol(-)(1) (based on measurements in water) and +16.5 +/- 2.0 cm(3) mol(-)(1) (in acetonitrile). For the Co(bipy)(3)(3+/2+) system, values are +12.7 +/- 1.4 cm(3) mol(-)(1) (in water) and +15.5 +/- 2.5 cm(3) mol(-)(1) (in acetonitrile). Using these experimentally determined intrinsic contributions, a simple structural model suggests that the intrinsic volume change for these reactions can be described using the change in effective volume of a sphere with radius close to that of the coordinating-atom-metal bond length. Electrostrictive volume changes for the 3+/2+ complex-ion couples are a function of solute size and coordinated ligands. For Ru(H(2)O)(6)(3+) and Fe(H(2)O)(6)(3+) reduction, volume behavior is significantly different from that of the other systems studied and can be rationalized in terms of possible H-bonding interactions with surrounding solvent which affect the electrostrictive volume changes but which are not available for the ammine and other complexes studied.