Mechanistic studies of metal independent phenylalanine hydroxylase from Chromobacterium violaceum

"Metal-free" phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) was prepared by extraction of copper from copper-containing CVPAH with dithiothreitol. These preparations were fully active, yet had a Cu/Enzyme ratio of 0.015. Reconstitution by extraneous copper was disproved by measuring a Cu/enzyme ratio of 0.09 in assay mixtures after finding nearly full activity, and by lack of inhibition by copper chelators. Copper and zinc were determined to have Kds of 0.48 and 0.85 {dollar}mu{dollar}M respectively, and both were inhibitors of CVPAH. No other metals were found in stoichiometric amounts. These results do not support a metal requirement for CVPAH and preclude any metal-oxo species as an intermediate in its reaction mechanism.; Two mutant forms of CVPAH were made by replacing histidines 138 and 143 with serines. Both mutants were completely inactive, yet both bound copper and phenylalanine. Electron spin echo envelope modulation of the mutant proteins exhibited a spectrum consistent with a single histidine in the coordination sphere of each mutant, rather than the two histidine ligation found in wild type CVPAH. This suggests that histidines 138 and 143 are the two equatorial histidine ligands in the copper inhibited CVPAH. The fact that CVPAH is inactivated by both mutation of these histidines and by formation of metal complexes with these histidines suggests that they may play a catalytic role in the CVPAH reaction.; The "metal-free" CVPAH reaction exhibited an NIH shift to the same degree as the iron-dependent PAH from rat liver. CVPAH was shown to hydroxylate alkyl carbons, producing the same ratio of alkyl to aromatic hydroxylation as RLPAH when 4-methyl-L-phenylalanine was the substrate. This indicates that the oxygenating intermediate is probably the same for each system. L-Cyclohexylalanine was hydroxylated by both RLPAH and CVPAH. This reaction was stereoselective, yielding {dollar}sim{dollar}90% cis-4-hydroxy-L-cyclohexylalanine with the remainder being the trans isomer no reaction was observed with 4-chloro-L-phenylalanine or L-tyrosine as a substrate. There is no pterin oxidase activity of CVPAH in the presence of peroxides. Fully uncoupled pterin oxidation was observed with L-norleucine as a CVPAH substrate. This reaction produced both {dollar}rm Hsb2Osb2{dollar} and 4a-hydoxy-tetrahydropterin. These results establish the precedence of a metal independent PAH enzyme that does not differ in any mechanistic parameter with the iron-dependent mammalian PAH. Therefore, the required iron in mammalian pterin dependent hydroxylases may not be involved in the formation of the activated oxygen intermediate.