Essential role of conserved arginine 160 in intramolecular electron transfer in human sulfite oxidase.
Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Changjian
C
Feng
Heather L
HL
Wilson
John K
JK
Hurley
James T
JT
Hazzard
Gordon
G
Tollin
K V
KV
Rajagopalan
John H
JH
Enemark
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D004579:Electron Transport; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry; D055672:Static Electricity
D001120:Arginine; D050862:Oxidoreductases Acting on Sulfur Group Donors