S Balasubramanian, RT Carr, CJ Bender, J Peisach, SJ Benkovic
Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) is known to bind an equivalent of divalent copper. The "metal-free" form of the protein is fully active, and Cu(II) is now shown to be an inhibitor of CVPAH rather than an activator of the enzyme [Carr, R. T., & Benkovic, S. J. (1994) Biochemistry 32, 14132-14138]. On the basis of amino acid sequence homology, the metal binding site may be related to those of rat liver PAH and other eukaryotic pterin-dependent hydroxylases, which require Fe(II) for activity. The conserved histidines at that site in CVPAH, histidines 138 and 143, were each mutated to serines. The mutant enzymes H138S and H143S were both catalytically inactive, but still able to bind Cu(II). Binding studies further demonstrated that both mutant enzymes still bind L-phenylalanine. Electron spin echo envelope modulation (ESEEM) studies on each of the mutants showed the presence of only a single copper-coordinating histidine, rather than the two histidine ligands suggested for the wild-type protein. This result supports a model in which Cu(II) is equatorially ligated to only two histidines in the Cu(II)-inhibited protein and allows us to unambiguously assign histidines 138 and 143 as these ligands. That the enzyme is inactive when these histidines are either bound with copper or when replaced with serines suggests that these histidines perform a catalytic function. Possible catalytic roles for these histidines in the hydroxylation mechanism of pterin-dependent monooxygenases are discussed along with potential future applications of the combination of ESEEM with site-directed mutagenesis.