Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.
The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.
Aldag
Caroline
C
Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
Gromov
Igor A
IA
García-Rubio
Inés
I
von Koenig
Konstanze
K
Schlichting
Ilme
I
Jaun
Bernhard
B
Hilvert
Donald
D
D019943:Amino Acid Substitution; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D015336:Molecular Probe Techniques; D010084:Oxidation-Reduction; D015202:Protein Engineering; D017279:Selenocysteine / Q000235:genetics*
D008024:Ligands; D017279:Selenocysteine; D006418:Heme; D019475:Camphor 5-Monooxygenase