Role of conserved tyrosine 343 in intramolecular electron transfer in human sulfite oxidase.
Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe(II)) and oxidized molybdenum (Mo(VI)) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k(cat). Steady-state kinetic analysis of the Y343F mutant showed an increase in K(m)(sulfite) and a decrease in k(cat) resulting in a 23-fold attenuation in the specificity constant k(cat)/K(m)(sulfite) at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.
Feng
Changjian
C
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Wilson
Heather L
HL
Hurley
John K
JK
Hazzard
James T
JT
Tollin
Gordon
G
Rajagopalan
K V
KV
Enemark
John H
JH
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D017124:Conserved Sequence; D004579:Electron Transport; D006418:Heme / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000494:pharmacology; D007700:Kinetics; D008982:Molybdenum / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010782:Photolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014443:Tyrosine
D006878:Hydroxides; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D008982:Molybdenum; D050862:Oxidoreductases Acting on Sulfur Group Donors