A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.

How Is Substrate Halogenation Triggered by the Vanadium Haloperoxidase from Curvularia inaequalis?

Vanadium haloperoxidases (VHPOs) are unique enzymes in biology that catalyze a challenging halogen transfer reaction and convert a strong aromatic C-H bond into C-X (X = Cl, Br, I) with the use of a vanadium cofactor and H2O2. The VHPO catalytic cycle starts with the conversion of hydrogen peroxide and halide (X = Cl, Br, I) into hypohalide on the vanadate cofactor, and the hypohalide subsequently reacts with a substrate. However, it is unclear whether the hypohalide is released from the enzyme or otherwise trapped within the enzyme structure for the halogenation of organic substrates. A substrate-binding pocket has never been identified for the VHPO enzyme, which questions the role of the protein in the overall reaction mechanism. Probing its role in the halogenation of small molecules will enable further engineering of the enzyme and expand its substrate scope and selectivity further for use in biotechnological applications as an environmentally benign alternative to current organic chemistry synthesis. Using a combined experimental and computational approach, we elucidate the role of the vanadium haloperoxidase protein in substrate halogenation. Activity studies show that binding of the substrate to the enzyme is essential for the reaction of the hypohalide with substrate. Stopped-flow measurements demonstrate that the rate-determining step is not dependent on substrate binding but partially on hypohalide formation. Using a combination of molecular mechanics (MM) and molecular dynamics (MD) simulations, the substrate binding area in the protein is identified and even though the selected substrates (methylphenylindole and 2-phenylindole) have limited hydrogen-bonding abilities, they are found to bind relatively strongly and remain stable in a binding tunnel. A subsequent analysis of the MD snapshots characterizes two small tunnels leading from the vanadate active site to the surface that could fit small molecules such as hypohalide, halide, and hydrogen peroxide. Density functional theory studies using electric field effects show that a polarized environment in a specific direction can substantially lower barriers for halogen transfer. A further analysis of the protein structure indeed shows a large dipole orientation in the substrate-binding pocket that could enable halogen transfer through an applied local electric field. These findings highlight the importance of the enzyme in catalyzing substrate halogenation by providing an optimal environment to lower the energy barrier for this challenging aromatic halide insertion reaction.

What Drives Radical Halogenation versus Hydroxylation in Mononuclear Nonheme Iron Complexes? A Combined Experimental and Computational Study.

Nonheme iron halogenases are unique enzymes in nature that selectively activate an aliphatic C-H bond of a substrate to convert it into C-X (X = Cl/Br, but not F/I). It is proposed that they generate an FeIII(OH)(X) intermediate in their catalytic cycle. The analogous FeIII(OH) intermediate in nonheme iron hydroxylases transfers OH• to give alcohol product, whereas the halogenases transfer X• to the carbon radical substrate. There remains significant debate regarding what factors control their remarkable selectivity of the halogenases. The reactivity of the complexes FeIII(BNPAPh2O)(OH)(X) (X = Cl, Br) with a secondary carbon radical (R•) is described. It is found that X• transfer occurs with a secondary carbon radical, as opposed to OH• transfer with tertiary radicals. Comprehensive computational studies involving density functional theory were carried out to examine the possible origins of this selectivity. The calculations reproduce the experimental findings, which indicate that halogen transfer is not observed for the tertiary radicals because of a nonproductive equilibrium that results from the endergonic nature of these reactions, despite a potentially lower reaction barrier for the halogenation pathway. In contrast, halogen transfer is favored for secondary carbon radicals, for which the halogenated product complex is thermodynamically more stable than the reactant complex. These results are rationalized by considering the relative strengths of the C-X bonds that are formed for tertiary versus secondary carbon centers. The computational analysis also shows that the reaction barrier for halogen transfer is significantly dependent on secondary coordination sphere effects, including steric and H-bonding interactions.

Proton-coupled electron transfer reactivities of electronically divergent heme superoxide intermediates: a kinetic, thermodynamic, and theoretical study.

Heme superoxides are one of the most versatile metallo-intermediates in biology, and they mediate a vast variety of oxidation and oxygenation reactions involving O2(g). Overall proton-coupled electron transfer (PCET) processes they facilitate may proceed via several different mechanistic pathways, attributes of which are not yet fully understood. Herein we present a detailed investigation into concerted PCET events of a series of geometrically similar, but electronically disparate synthetic heme superoxide mimics, where unprecedented, PCET feasibility-determining electronic effects of the heme center have been identified. These electronic factors firmly modulate both thermodynamic and kinetic parameters that are central to PCET, as supported by our experimental and theoretical observations. Consistently, the most electron-deficient superoxide adduct shows the strongest driving force for PCET, whereas the most electron-rich system remains unreactive. The pivotal role of these findings in understanding significant heme systems in biology, as well as in alternative energy applications is also discussed.

Mechanistic Investigation of Oxygen Rebound in a Mononuclear Nonheme Iron Complex.

An iron(III) methoxide complex reacts with para-substituted triarylmethyl radicals to give iron(II) and methoxyether products. Second-order rate constants for the radical derivatives were obtained. Hammett and Marcus plots suggest the radical transfer reactions proceed via a concerted process. Calculations support the concerted nature of these reactions involving a single transition state with no initial charge transfer. These findings have implications for the radical "rebound" step invoked in nonheme iron oxygenases, halogenases, and related synthetic catalysts.