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. 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-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.

Figure 1

(A) X-ray crystal structure of the catalytic core of peptidylglycine α-hydroxylating monooxygenase (PDB: 1PHM). (B) The active site is enlarged to show the ligands coordinating to Cu_M (H242, H244, and M314) and Cu_H (H107, H108, and H172) and the conserved tyrosine (Y79 in PHM and Y216 in TβM) that is the focus of this study. Residue numbers are shown for PHM (without parentheses) and TβM (with parentheses).

Scheme 1. Proposed Mechanism for TβM

This is a generic mechanism in which the ligands to copper and the movement of solvent protons are left ambiguous.

Figure 2

Solvent isotope effects for Tyr216Ala TβM. (A,B) pH profiles in H₂O (blue) vs D₂O (red). (C,D) Isotope effects on ^Dk_cat and ^D(k_cat/K_m), respectively. Error bars are presented as standard error, or 68% confidence interval.

Figure 3

Apparent rate constant and KIE of Tyr216Trp TβM at various concentrations of tyramine, (A,B) at pL 6.0 and (C,D) at pL 7.5. Error bars are presented as standard error, or 68% confidence interval.

Figure 4

Proton inventory experiment of Tyr216Ala TβM at pL 7.5. The solid lines represent a single PT (solid line), two-site transfer with the same KIE (dashed line), and an infinite site transfer model (dotted line). Errors are presented as ±1σ, or 68% confidence interval.

Scheme 2. Proposed Mechanism for the PCET Step

Figure 5

Plot of fitted temperature dependence of ET rate. (A) The experimental data cannot be fit from enthalpic terms alone. (B) Incorporating an entropic term into either ΔG⁰ or λ (Table 3) allows the data to be fit quite well. The curves in panels A and B are equivalent for ΔG⁰ = 0, 5, or 10 kcal/mol.