A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu(M) site of tyramine beta-monooxygenase (TbetaM). The methionine ligand at Cu(M) is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TbetaM mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type TbetaM in the oxidized enzyme forms. EPR spectroscopy suggests that the Cu(II) coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys TbetaM. Met471Cys TbetaM provides the first example of an active mutant directed at the Cu(M) site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.

Background: The function of CsoR/RcnR-like protein InrS was unknown.Results: InrS and CsoR metal complexes have similar spectra, but InrS senses nickel not copper.Conclusion: InrS detects cytosolic nickel with tighter KD than the other metal sensors of the cell.Significance: InrS might be exploited to optimize hydrogenase Ni(II) supply, and this study shows how metal specificity can be a shared function of a set of metal sensors.

Tyramine ␤-monooxygenase (T␤M) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of T␤M for comparison with the homologous mammalian enzymes, dopamine ␤-monooxygenase and peptidylglycine ␣-hydroxylating monooxygenase. A new and distinctive feature of T␤M is very strong substrate inhibition that is dependent on the level of the co-substrate, O 2 , and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of T␤M, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of T␤M is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.The copper hydroxylases are a unique class of enzymes that are found in eukaryotes and play a critical role in the biosynthesis of neurotransmitters and hormones. The most studied enzymes in this family are peptidylglycine ␣-hydroxylating monooxygenase (PHM) 3 and dopamine ␤-monooxygenase (D␤M) (1). PHM catalyzes the conversion of C-terminal glycine-extended peptides to their ␣-hydroxylated products, the first step in the amidation of peptide hormones, required for a range of biological activities (2). D␤M catalyzes the hydroxylation of dopamine to yield norepinephrine and, thus, is vital for the regulation of these neurotransmitters (3-5). More recently, a third member of this enzyme family was identified, tyramine ␤-monooxygenase (6). T␤M is the insect homolog of D␤M, sharing 39% identity and 55% similarity with the mammalian enzyme. T␤M similarly catalyzes the hydroxylation of tyramine at the ␤-carbon position (Scheme 1). Although tyramine plays no role in mammalian physiology, the product of T␤M, octopamine, has been shown to act as a neurotransmitter in invertebrates, regulating physiological functions such as neuromuscular transmission, behavioral development, and ovulation (7-9). One advantage to studies with T␤M is the much more facile expression system for this enzyme in relation to D␤M (10), which makes it possible to pursue structure/function relationships. In this paper, we report a detailed analysis of the kinetic behavior of the wild type T␤M, an essential first step in understanding this complex enzyme system.All three of the copper hydroxylases employ two noncoupled, mononuclear Cu centers for oxygenase activity, termed Cu M and Cu H . Structural information pertaining to the active site of the enzymes is derived primarily from the crystal structure for PHM and extended X-ray absorption fine structure studies with both D␤M and PHM (11-13). Each copper site assumes a unique coordination environment and a distinct mechanistic function. Cu M serves as the site of dioxygen binding and activation, whereas the Cu H site functions as an electron transfer site in the reac...

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2)2–CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [CuM(His)2S(Met)–CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)–S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu–S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the CuM center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-010-0677-3) contains supplementary material, which is available to authorized users.