Solvent and Temperature Probes of the Long-Range Electron-Transfer Step in Tyramine β-Monooxygenase: Demonstration of a Long-Range Proton-Coupled Electron-Transfer Mechanism

Abstract

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. Biochemistry2013, 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-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.

The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution

“…Finally, ascorbic acid binds to the substrate intermediate, triggering the release of the product. However, although a recent study on TBH has demonstrated a proton-coupled long-range electron transfer mechanism ( 41 ), one of the more difficult things to appreciate is how the necessary electron transfer proceeds over a distance of more than 10 Å with the lack of domain movement ( 25 , 26 ). The DBH structure indicates that maybe domain movement places the two copper sites close together during the electron transfer step.…”

Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase

“…An alternative hypothesis for the ET pathway of the catalytic electron has recently been suggested from studies on the TBM system. Unlike the TBM Y216W/I variants, the TBM Y216A mutant shows rate-limiting electron transfer (27), which has allowed the deuterium solvent isotope effect on this rate constant to be determined (40). The size of this isotope effect and its T-dependence indicate a large entropic contribution to the driving force, suggesting solvent reorganization as a critical step in the catalytic ET process.…”

“…A number of proposals for the catalytic ET step have been advanced. These have included superoxide channeling (38); a “substrate-mediated” pathway involving H108, Q170, a water molecule, and the peptide substrate; direct interaction of the Cu(I)H with the substrate radical via domain movement (39); and transfer across the inter-site cavity via an ordered array of water molecules, perhaps assisted by Y79 (26, 27, 40). However, these mechanisms do not account for all of the available data.…”

Moving Through Barriers in Science and Life

“…An exciting collaboration between Dale Edmondson at Emory University and Mitch Brenner in my laboratory led to the important discovery that DβM requires two uncoupled copper ions per active site to function (12); this finding was followed by single-turnover, rapid mixing experiments to show that both of the (prereduced) copper centers undergo rapid reoxidation in a single phase that correlates with the time constant for the formation of norepinephrine from dopamine (13). The presence of two uncoupled copper centers was later confirmed by X-ray crystallography (14), introducing the new challenge of understanding how spatially distant copper centers separated by solvent water molecules are capable of supporting a highly controlled oxygen insertion into dopamine (15,16), in the absence of any of the uncoupling reactions (17) commonly seen in monooxygenases. Our most recent proposal of a long-range proton-coupled electron transfer (PCET) ( 16) is relevant to many biological processes and has emerged as a highly active research area at the interface of theory and experiment.…”

“…As with hydrogen and its three isotopes (H, D, and T), oxygen also offers three isotopes ( 16 O, 17 O, and 18 O) as mechanistic probes. Although our primary focus was, over many years, the comparison of 16 O with 18 O, an early study made use of all three oxygen isotopes in the context of looking for alternative explanations to the huge (nonclassical) KIEs first seen in the lipoxygenase reaction.…”