On the active site of old yellow enzyme - Role of histidine 191 and asparagine 194

Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500 -800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxy gen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron accepters to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A . (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phen olic ligands, stabilizing the anionic form involved in charge-transfer inte raction with the FMN prosthetic group. His-191 and Asn-194 are also predict ed to interact with the nicotinamide ring of NADPH in the active site. Muta tions of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H , were made of OYE1. It was not possible to isolate the N191H mutant enzyme , but the other two mutant forms had the expected effect on phenolic ligand binding, i,e, decreased binding affinity and decreased charge-transfer abs orbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. Th e double mutant enzyme had an increased rate constant for reduction by NADP H, but the reduction rate constant with NADH was lower by a factor of 15. T he reactivity of OYE1 and the mutant enzymes with oxygen was similar, but t he reactivity of 2-cyclohexenone was greatly decreased by the mutations. Th e crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme.