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Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.

ABSTRACT: Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5-OO(H) bond at 100 K, thus releasing O2 in situ, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site.

SUBMITTER: Bui S

PROVIDER: S-EPMC4502973 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.

Bui Soi S von Stetten David D Jambrina Pablo G PG Prangé Thierry T Colloc'h Nathalie N de Sanctis Daniele D Royant Antoine A Rosta Edina E Steiner Roberto A RA

Angewandte Chemie (International ed. in English) 20141014 50

Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide inte ...[more]

PMID: 25314114

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Project description:The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP+ undergoes a partial hydride transfer reaction, while E·GMP·NADP+ undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH3/NH4+ has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.

Dataset Information

Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.

Publications

Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.

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