Solventand Temperature Probes of the Long-Range Electron-Transfer Step inTyramine β-Monooxygenase: Demonstration of a Long-RangeProton-Coupled Electron-Transfer Mechanism

摘要

Tyramine β-monooxygenase (TβM) 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 TβM, 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 TβM 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-rangeET step, a temperature dependence study was also pursued. The smallenthalpic barrier of ET (Ea = 3.6 kcal/mol)implicates a significant entropic barrier, which is attributed tothe requirement for extensive rearrangement of the inter-copper environmentduring PCET catalyzed by the Tyr216Ala variant. The data lead to theproposal of a distinct inter-domain pathway for PCET in the dinuclearcopper monooxygenases.