Identification of core active site residues of the sulfur oxygenase reductase from Acidianus ambivalens by site-directed mutagenesis.
The sulfur oxygenase reductase (SOR) is the initial enzyme in the sulfur oxidation pathway of Acidianus ambivalens. The SOR is composed of 308 aa residues, three of which are cysteines, and contains a mononuclear non-heme iron site. Mutations of the suspected iron-binding residues H86, H90 and E114 to alanine resulted in inactive enzyme with no iron incorporated, whereas an E114D mutant showed 1% of wild type activity. The mutation of C31 to alanine and serine caused inactivity of the enzyme, however, the iron content was the same as in the wild type. C101A, C104S/A, and C101/104S/A double mutants caused a decrease in specific activity to 10-43% of the wild type while the C101S mutant showed only 1% activity of the wild type. The drop in activity of the C101S and E114D mutants was accompanied with a proportional decrease in iron content. In all cases the oxygenase and reductase partial reactions were equally affected. It was concluded that the Fe site with H86, H90 and E114 as ligands and C31 constitute the core active site whereas C101 and C104 optimize reaction conditions.
Urich
Tim
T
Institute of Microbiology and Genetics, Darmstadt University of Technology, Schnittspahnstrasse 10, 64287 Darmstadt, Germany.
Kroke
Anja
A
Bauer
Christian
C
Seyfarth
Kerstin
K
Reuff
Muriel
M
Kletzin
Arnulf
A
D041541:Acidianus / Q000201:enzymology* / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine; D007501:Iron; D016296:Mutagenesis; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors
D019843:Archaeal Proteins; D007501:Iron; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C058771:soR protein, Acidianus ambivalens; D003545:Cysteine