SELECTIVE CYSTEINE-]SERINE REPLACEMENTS IN P-HYDROXYBENZOATE HYDROXYLASE FROM PSEUDOMONAS-FLUORESCENS ALLOW THE UNAMBIGUOUS ASSIGNMENT OF CYS211 AS THE SITE OF MODIFICATION BY SPIN-LABELED P-CHLOROMERCURIBENZOATE

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains fi ve sulfhydryl groups per subunit. Cysteine --> serine replacements sho w that the thiols are not essential for catalysis. The increased disso ciation constant for FAD in mutant Cys158Ser suggests that Cys158 is i mportant for the solvation of the pyrophosphate moiety of the prosthet ic group. Wild-type p-hydroxybenzoate hydroxylase is rapidly inactivat ed by mercurial compounds. Inactivation by a spin-labeled derivative o f p-chloromercuribenzoate is fully abolished in mutant Cys211Ser. Inco rporation of the spin label in the other Cys --> Ser mutants strongly impairs substrate binding without affecting the catalytic properties o f the FAD. The results are discussed with respect to previous tentativ e assignments from chemical modification studies and in light of the 3 -D structure of the enzyme-substrate complex.