Intramolecular electron transfer (IET) between the molybdenum and heme centers of vertebrate sulfite oxidase (SO) is proposed to be a key step in the catalytic cycle of the enzyme. However, the X-ray crystallographic distance between these centers, RMoFe = 32.3 Å, appears to be too long for the rapid IET rates observed in liquid solution. The Mo and heme domains are linked by a flexible tether, and it has been proposed that dynamic interdomain motion brings the two metal centers closer together and thereby facilitates rapid IET. To date there have been no direct distance measurements for SO in solution that would support or contradict this model. In this work, pulsed electron-electron double resonance (ELDOR) and relaxation induced dipolar modulation enhancement (RIDME) techniques were used to obtain information about RMoFe in the Mo(V)Fe(III) state of wild type recombinant human SO in frozen glassy solution. Surprisingly, the data obtained suggest a fixed structure with RMoFe = 32 Å, similar to that determined by X-ray crystallography for chicken SO, although the orientation of the RMoFe radius-vector with respect to the heme center was found to be somewhat different. The implications of these findings for the flexible tether model are discussed.

Human sulfite oxidase (hSO), an essential molybdoheme enzyme, catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic cycle includes two, one-electron intramolecular electron transfers (IET) between the molybdenum (Mo) and the heme domains. Rapid IET rates are ascribed to conformational changes that bring the two domains into close proximity to one another. Previous studies of hSO have focused on the roles of conserved residues near the Mo active site and on the tether that links the two domains. Here four aromatic surface residues on the heme domain (phenylalanine 57 (F57), phenylalanine 79 (F79), tyrosine 83 (Y83), and histidine 90 (H90)) have been mutated, and their involvement in IET rates, the heme midpoint potential, and the catalytic activity of hSO have been investigated using laser flash photolysis, spectroelectrochemistry, and steady-state kinetics, respectively. The results indicate that the size and hydrophobicity of F57 play an important role in modulating the heme potential and that F57 also affects the IET rates. The data also suggest that important interactions of H90 with a heme propionate group destabilize the Fe(III) state of the heme. The positive charge on H90 at pH ≤ 7.0 may decrease the electrostatic interaction between the Mo and heme domains, thereby decreasing the IET rates of wt hSO at low pH. Lastly, mutations of F79 and Y83, which are located on the surface of the heme domain, but not in direct contact with the heme or the propionate groups, have little effect on either IET or the heme potential.

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