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 five sulfhydryl groups per subunit. Cysteine-->serine replacements show that the thiols are not essential for catalysis. The increased dissociation constant for FAD in mutant Cys158Ser suggests that Cys158 is important for the solvation of the pyrophosphate moiety of the prosthetic group. Wild-type p-hydroxybenzoate hydroxylase is rapidly inactivated by mercurial compounds. Inactivation by a spin-labeled derivative of p-chloromercuribenzoate is fully abolished in mutant Cys211Ser. Incorporation of the spin label in the other Cys-->Ser mutants strongly impairs substrate binding without affecting the catalytic properties of the FAD. The results are discussed with respect to previous tentative assignments from chemical modification studies and in light of the 3-D structure of the enzyme-substrate complex.