Intramolecular electron transfer in sulfite-oxidizing enzymes: elucidating the role of a conserved active site arginine.
All reported sulfite-oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. To improve our understanding of the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions R55Q and R55M as well as the positively charged substitution R55K in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for improving our understanding of residue function in IET without the added complexity of the interdomain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) exhibited increased IET rate constants relative to that of the wild type (3-4-fold) when studied by laser flash photolysis. The gain in function observed in SDH(R55Q) and SDH(R55M) suggests that the reduction in the level of IET seen in HSO(R160Q) is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the interdomain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDH(WT), SDH(R55M), and SDH(R55Q) show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (K(eq)) and rate constants (k(et)) of both variants with respect to the SDH(WT) enzyme. SDH(WT) has a k(et) that is independent of pH and a K(eq) that increases as pH decreases; on the other hand, both SDH(R55M) and SDH(R55Q) have a k(et) that increases as pH decreases, and SDH(R55M) has a K(eq) that is pH-independent. IET in the SDH(R55Q) variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from that of SDH(WT), but which also occurs in HSO. IET in SDH(R55K) is slower than in SDH(WT). A new analysis of the possible mechanistic pathways for sulfite-oxidizing enzymes is presented and related to available kinetic and EPR results for these enzymes.
Emesh
Safia
S
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
Rapson
Trevor D
TD
Rajapakshe
Asha
A
Kappler
Ulrike
U
Bernhardt
Paul V
PV
Tollin
Gordon
G
Enemark
John H
JH
D019943:Amino Acid Substitution / Q000502:physiology; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000502:physiology*; D004579:Electron Transport; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010782:Photolysis; D020560:Proteobacteria / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013431:Sulfates / Q000737:chemistry
D011994:Recombinant Proteins; D013431:Sulfates; D001120:Arginine; D050862:Oxidoreductases Acting on Sulfur Group Donors; C503599:SUOX protein, human