Intramolecular electron transfer in sulfite-oxidizing enzymes: Elucidating the role of a conserved active site arginine

Emesh, Safia, Rapson, Trevor D., Rajapakshe, Asha, Kappler, Ulrike, Bernhardt, Paul V., Tollin, Gordon and Enemark, John H. (2009) Intramolecular electron transfer in sulfite-oxidizing enzymes: Elucidating the role of a conserved active site arginine. Biochemistry, 48 10: 2156-2163. doi:10.1021/bi801553q

Author Emesh, Safia
Rapson, Trevor D.
Rajapakshe, Asha
Kappler, Ulrike
Bernhardt, Paul V.
Tollin, Gordon
Enemark, John H.
Title Intramolecular electron transfer in sulfite-oxidizing enzymes: Elucidating the role of a conserved active site arginine
Journal name Biochemistry   Check publisher's open access policy
ISSN 0006-2960
Publication date 2009-02-02
Year available 2009
Sub-type Article (original research)
DOI 10.1021/bi801553q
Volume 48
Issue 10
Start page 2156
End page 2163
Total pages 8
Editor Richard N. Armstrong
Place of publication United States
Publisher American Chemical Society
Collection year 2010
Language eng
Subject C1
060107 Enzymes
970106 Expanding Knowledge in the Biological Sciences
Formatted abstract
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 SDHR55Q and SDHR55M suggests that the reduction in the level of IET seen in HSOR160Q 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 SDHWT, SDHR55M, and SDHR55Q show that the arginine substitution also alters the behavior of the Mo−heme IET equilibrium (Keq) and rate constants (ket) of both variants with respect to the SDHWT enzyme. SDHWT has a ket that is independent of pH and a Keq that increases as pH decreases; on the other hand, both SDHR55M and SDHR55Q have a ket that increases as pH decreases, and SDHR55M has a Keq that is pH-independent. IET in the SDHR55Q variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from that of SDHWT, but which also occurs in HSO. IET in SDHR55K is slower than in SDHWT. 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.
Copyright © 2009 American Chemical Society
Q-Index Code C1
Q-Index Status Confirmed Code

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
School of Chemistry and Molecular Biosciences
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 24 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 23 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Thu, 03 Sep 2009, 08:32:45 EST by Mr Andrew Martlew on behalf of School of Chemistry & Molecular Biosciences