Inactivation of Met471Cys tyramine β-monooxygenase results from site-specific cysteic acid formation.

Tyramine β-monooxygenase (TβM) belongs to a small class of eukaryotic copper-, ascorbate-, and O2-dependent enzymes that includes dopamine β-monooxygenase (DβM) and peptidylglycine α-hydroxylating monooxygenase (PHM) (1-3). TβM is the insect homologue of DβM and plays a neuroregulatory role in insects by catalyzing the β-hydroxylation of tyramine to yield octopamine (eq. 1) (4-6). (1) Studies indicate that TβM expression occurs in octopaminergic neurons (5,6). A significant amount of spectroscopic and kinetic data collected for these enzymes has demonstrated a high conservation of mechanism (7-14). Structural information about TβM is inferred from primary amino acid sequence homology and the X-ray structures solved for PHM (3,15-17). TβM is assumed to use two solvent-exposed, non-coupled mononuclear copper centers, denoted CuM and CuH and separated by ~ 11 A (Figure 1), to incorporate an oxygen atom into the phenethylamine side chain of tyramine (3,14), eq. 1. O2 activation and substrate hydroxylation occur exclusively at the CuM domain, which is coordinated by two histidines and a Met residue (17,18). CuH, which is ligated by three histidine residues, serves as an electron reservoir to supply the second electron required for insertion of oxygen into the C–H bond by long-range intramolecular electron transfer (7). The combination of structural, kinetic, biochemical, and theoretical evidence supports the formation of a CuM-superoxo intermediate as the active oxygen species responsible for C–H activation at the CuM site (1,17,19-22); by contrast, the precise timing and pathway for electron transfer from CuH to CuM remain under active debate (19,20,22). Figure 1 (Recreated from PDB: 1PHM) (15). The ligands at CuH (His107, His108 and His172) and to CuM (His242, His244, Met314) of PHM. Met471 in TβM corresponds to Met314 in PHM. The Met residue at the CuM domain appears to be a critical component for catalysis by these enzymes, but the exact function of this residue is not known. The ability of the CuM-Met ligand to move into the coordination sphere upon reduction of the copper domains is supported by spectroscopic and crystallographic analyses (2,7,9-11,15,23). Extended X-ray absorption fine structure (EXAFS) for the Cu(II) state of PHM, DβM and TβM indicates an average of 2.5 nitrogen (His) and 1.5 oxygen or nitrogen ligands per copper at 1.97 A (2,7,10,11,23) and a weak sulfur ligand detected at 2.71 A, in agreement with the PHM X-ray crystal structure (15). Enzyme reduction leads to a shorter Met to copper bond distance: 2.27 A in PHM, 2.25 A in DβM, and 2.24 A in TβM (2,10,11,23). Importantly, despite the active site solvent exposure of these enzymes and the requirement for cycling between Cu(I) and Cu(II) during each catalytic cycle, the consumption of O2 and the generation of hydroxylated product are tightly coupled. Neither the use of a very slow substrate with DβM (19) or site-specific mutation in PHM (His172Ala) (20), both of which diminished kcat by nearly three orders of magnitude, has any impact on the 1:1 ratio for O2 uptake and product formation. The aggregate data implicate a dynamic role for the Met ligand to CuM that regulates the equilibrium for generating a copper-superoxo species and prevents the diffusional loss of activated oxygen species during turnover (1,19,22). Site-directed mutagenesis has been used extensively to understand the significance of the Met ligand to CuM. Studies with PHM in which Met314 was substituted with Ile, His, Asp, and Cys, reported that the hydroxylation of glycine-extended peptides was undetectable in spent media (2,24), whereas a low activity has been reported for Met314His (25). The X-ray structure of the Met314Ile PHM variant was found to lead to a significant disorder at the CuH domain, implicating communication between the ligands at distal metal sites (26). Recently, the analogous Met ligand in TβM was mutated to a Cys, His, and Asp, and only the Met471Cys retained measureable activity. Electron paramagnetic resonance (EPR) characterization of the Cu(II) forms of WT and Met471 variants have shown identical properties. Interestingly, Met471Cys TβM was unable to fully convert substrate to product under any conditions examined. This result led to the proposal of a secondary inactivation pathway during turnover (27). XAS/EXAFS experiments were carried out on the series of Met471 variants, to examine how changes in structure relate to the observed altered reactivity, and these showed no binding of His and Asp to Cu(I)M and only a very weak/small interaction between Cys and the metal site (23). Herein, we demonstrate that the replacement of Met471 by Cys generates an enzyme form that undergoes directed oxidation at position 471. This is attributed to the poor coordination between Cys471 and Cu(I)M, and not to a substrate-dependent inactivation as previously suggested (27).