Kinetic and Thermodynamic Effects of Mutations of Human Sulfite Oxidase

Animal SOs possess two redox-active domains and are ideal for studying IET in relation to the overall conformation of the protein and the molecular dynamics of the motion of the two domains relative to one another. The procedures for preparing and purifying recombinant hSO [34, 44] have been further optimized in our laboratory [4, 5, 29], so that a wide range of mutant proteins in 50–100 mg quantities can be prepared for comprehensive kinetic, mechanistic, and spectroscopic studies. The ready accessibility of recombinant hSO has made it possible to investigate the effects of modifying amino acid residues of interest to better understand the long range IET process in hSO and to obtain insight into biochemical basis of pathological mutations of hSO. Experimental and theoretical studies agree that domain movement is necessary for SO to achieve a productive conformation during electron transfer, and that the length and composition of the interdomain tether is an important factor in the rates of IET. However, the interactions such as hydrogen bonding and hydrophobic effects involved in mediating the correct mutual orientation and the distance following the initial orientation are unknown. Studies of site-specific mutants reveal a great deal about the IET complex in hSO. Mutating several aromatic residues that extend from the pterin to the surface of the protein suggest that alternative ET pathways may be in operation in hSO. The profound effects of mutating residues surrounding the Mo active site (e. g. R160Q and Y343F) have been demonstrated [28, 45], and extension to mutations of other positively charged residues surrounding the active site, such as Lys200, is warranted. Finally, all of the discussions presented here and elsewhere for IET in hSO rest upon the single structure of intact native cSO. It is clear that X-ray crystal structures of hSO and various mutants are needed to better understand the unexpected properties of this fascinating biomedically important protein.