Catalytic M Center of Copper Monooxygenases Probed by Rational Design. Effects of Selenomethionine and Histidine Substitution on Structure and Reactivity
| Autor: | Katherine B. Alwan, Evan F. Welch, Ninian J. Blackburn |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Stereochemistry
Peptidylglycine monooxygenase Ascorbic Acid Biochemistry Article Mixed Function Oxygenases Hydroxylation chemistry.chemical_compound Copper Transport Proteins Coordination Complexes Multienzyme Complexes Catalytic Domain Escherichia coli Histidine Reactivity (chemistry) Selenomethionine Ligand Escherichia coli Proteins Rational design Substrate (chemistry) Monooxygenase Oxygen Amino Acid Substitution chemistry Mutation Oxidation-Reduction Copper |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b00823 |
| Popis: | The M-centers of the mononuclear monooxygenases peptidylglycine monooxygenase (PHM) and dopamine β-monooxygenase (DBM) bind and activate dioxygen on route to substrate hydroxylation. Recently we reported the rational design of a protein-based model wherein the CusF metallochaperone was repurposed via a His to Met mutation to act as a structural and spectroscopic biomimic. The PHM M-site exhibits a number of unusual attributes including a His(2)Met ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N(3)(−), and complete coupling of oxygen reduction to substrate hydroxylation even at extremely low turnover rates. In particular, mutation of the Met ligand to His completely eliminates catalytic activity despite the propensity of Cu(I)-His(3) centers to bind and activate dioxygen in other metalloenzyme systems. Here we further develop the CusF-based model to explore methionine variants where Met is replaced by selenomethionine (SeM) and histidine. We examine the effects on coordinate structure and exogenous ligand binding via XAS and EPR and probe the consequences of mutations on redox chemistry via studies on the reduction by ascorbate, and oxidation via molecular oxygen. The M-site model is 3-coordinate in the Cu(I) state and binds CO to form a 4-coordinate carbonyl. In the oxidized forms the coordination changes to tetragonal 5-coordinate with a long axial Met ligand which like the enzymes is undetectable at either the Cu or Se K edges. The EXAFS data at the Se K-edge of the SeM variant provides unique information on the nature of the Cu-methionine bond which is likewise weak and fluxional. Kinetic studies document sluggish reactivity of the Cu(I) complexes with molecular oxygen and rapid rates of reduction of the Cu(II) complexes by ascorbate, indicating a remarkable stability of the Cu(I) state in all three derivatives. The results show little difference between the Met ligand and its SeM and His congeners and suggest that the Met contributes to catalysis in ways that are more complex than simple perturbation of the redox chemistry. Overall the results stimulate critical re-examination of the canonical reaction mechanisms of the mononuclear copper monooxygenases. |
| Databáze: | OpenAIRE |
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