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Low potential enzymatic hydride transfer via highly cooperative and inversely functionalized flavin cofactors.

ABSTRACT: Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to -340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°' = -493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.

SUBMITTER: Willistein M

PROVIDER: S-EPMC6502838 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Low potential enzymatic hydride transfer via highly cooperative and inversely functionalized flavin cofactors.

Willistein Max M Bechtel Dominique F DF Müller Christina S CS Demmer Ulrike U Heimann Larissa L Kayastha Kanwal K Schünemann Volker V Pierik Antonio J AJ Ullmann G Matthias GM Ermler Ulrich U Boll Matthias M

Nature communications 20190506 1

Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to -340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°' = -493 ...[more]

PMID: 31061390

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Project description:Transient oxidation-reduction through hydride transfer with tightly bound NAD coenzyme is used by a large class of sugar nucleotide epimerases to promote configurational inversion of carbon stereocenters in carbohydrate substrates. A requirement for the epimerases to coordinate hydride abstraction and re-addition with substrate rotation in the binding pocket poses a challenge for dynamical protein conformational selection linked to enzyme catalysis. Here, we studied the thermophilic C2 epimerase from Thermodesulfatator atlanticus (TaCPa2E) in combination with a slow CDP-glucose substrate (k cat ≈ 1.0 min-1; 60 °C) to explore the sensitivity of the enzymatic hydride transfer toward environmental fluctuations affected by temperature (20-80 °C). We determined noncompetitive primary kinetic isotope effects (KIE) due to 2H at the glucose C2 and showed that a normal KIE on the k cat (D k cat) reflects isotope sensitivity of the hydrogen abstraction to enzyme-NAD+ in a rate-limiting transient oxidation. The D k cat peaked at 40 °C was 6.1 and decreased to 2.1 at low (20 °C) and 3.3 at high temperature (80 °C). The temperature profiles for k cat with the 1H and 2H substrate showed a decrease in the rate below a dynamically important breakpoint (∼40 °C), suggesting an equilibrium shift to an impaired conformational landscape relevant for catalysis in the low-temperature region. Full Marcus-like model fits of the rate and KIE profiles provided evidence for a high-temperature reaction via low-frequency conformational sampling associated with a broad distribution of hydride donor-acceptor distances (long-distance population centered at 3.31 ± 0.02 Å), only poorly suitable for quantum mechanical tunneling. Collectively, dynamical characteristics of TaCPa2E-catalyzed hydride transfer during transient oxidation of CDP-glucose reveal important analogies to mechanistically simpler enzymes such as alcohol dehydrogenase and dihydrofolate reductase. A loose-fit substrate (in TaCPa2E) resembles structural variants of these enzymes by extensive dynamical sampling to balance conformational flexibility and catalytic efficiency.

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Low potential enzymatic hydride transfer via highly cooperative and inversely functionalized flavin cofactors.

Publications

Low potential enzymatic hydride transfer via highly cooperative and inversely functionalized flavin cofactors.

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