Solvent and Temperature Probes of the Long-Range Electron-Transfer Step in Tyramine ß-Monooxygenase: Demonstration of a Long-Range Proton-Coupled Electron-Transfer Mechanism.
J Am Chem Soc
; 137(17): 5720-9, 2015 May 06.
Article
en En
| MEDLINE
| ID: mdl-25919134
Tyramine ß-monooxygenase (TßM) belongs to a family of physiologically important dinuclear copper monooxygenases that function with a solvent-exposed active site. To accomplish each enzymatic turnover, an electron transfer (ET) must occur between two solvent-separated copper centers. In wild-type TßM, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. Biochemistry 2013, 52, 1179] that the Tyr216Ala variant of TßM leads to rate-limiting ET. In this study, we present a pH-rate profile study of Tyr216Ala, together with deuterium oxide solvent kinetic isotope effects (KIEs). A solvent KIE of 2 on kcat is found in a region where kcat is pH/pD independent. As a control, the variant Tyr216Trp, for which ET is not rate determining, displays a solvent KIE of unity. We conclude, therefore, that the observed solvent KIE arises from the rate-limiting ET step in the Tyr216Ala variant, and show how small solvent KIEs (ca. 2) can be fully accommodated from equilibrium effects within the Marcus equation. To gain insight into the role of the enzyme in the long-range ET step, a temperature dependence study was also pursued. The small enthalpic barrier of ET (Ea = 3.6 kcal/mol) implicates a significant entropic barrier, which is attributed to the requirement for extensive rearrangement of the inter-copper environment during PCET catalyzed by the Tyr216Ala variant. The data lead to the proposal of a distinct inter-domain pathway for PCET in the dinuclear copper monooxygenases.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Protones
/
Temperatura
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Proteínas de Drosophila
/
Oxigenasas de Función Mixta
Límite:
Animals
Idioma:
En
Revista:
J Am Chem Soc
Año:
2015
Tipo del documento:
Article
Pais de publicación:
Estados Unidos