Protonation State of MnFe and FeFe Cofactors in a Ligand-Binding Oxidase Revealed by X-ray Absorption, Emission, and Vibrational Spectroscopy and QM/MM Calculations

Abstract

Enzymes with a dimetal-carboxylate cofactor catalyze reactions among the top challenges in chemistry such as methane and dioxygen (O) activation. Recently described proteins bind a manganese-iron cofactor (MnFe) instead of the classical diiron cofactor (FeFe). Determination of atomic-level differences of homo- versus hetero-bimetallic cofactors is crucial to understand their diverse redox reactions. We studied a ligand-binding oxidase from the bacterium Geobacillus kaustophilus (R2lox) loaded with a FeFe or MnFe cofactor, which catalyzes O reduction and an unusual tyrosine-valine ether cross-link formation, as revealed by X-ray crystallography. Advanced X-ray absorption, emission, and vibrational spectroscopy methods and quantum chemical and molecular mechanics calculations provided relative Mn/Fe contents, X-ray photoreduction kinetics, metal-ligand bond lengths, metal-metal distances, metal oxidation states, spin configurations, valence-level degeneracy, molecular orbital composition, nuclear quadrupole splitting energies, and vibrational normal modes for both cofactors. A protonation state with an axial water (HO) ligand at Mn or Fe in binding site 1 and a metal-bridging hydroxo group (μOH) in a hydrogen-bonded network is assigned. Our comprehensive picture of the molecular, electronic, and dynamic properties of the cofactors highlights reorientation of the unique axis along the Mn-OH bond for the Mn1(III) Jahn-Teller ion but along the Fe-μOH bond for the octahedral Fe1(III). This likely corresponds to a more positive redox potential of the Mn(III)Fe(III) cofactor and higher proton affinity of its μOH group. Refined model structures for the Mn(III)Fe(III) and Fe(III)Fe(III) cofactors are presented. Implications of our findings for the site-specific metalation of R2lox and performance of the O reduction and cross-link formation reactions are discussed.

Ether cross-link formation in the R2-like ligand-binding oxidase

“…The Mn III /Fe III and Fe III /Fe III cofactors in R2lox have different electronic structures, with the unique axis oriented along the Mn–water bond for the Mn III ion, but along the Fe–μ–OH bond for the Fe III ion in site 1, which likely leads to the Mn/Fe cofactor having a slightly more positive redox potential [26, 27]. The different electronic structures and redox potentials of the two cofactors are expected to impact their catalytic potential and lead to different reactivities.…”

“…Mn1 also has a free coordination site. Y175, which usually forms a hydrogen bond with the water molecule bound to Mn1 [24, 27], is rotated in reduced V72L-R2lox and may instead form a hydrogen bond with the unliganded carboxyl oxygen of the N-terminal metal ligand E69, the other hydrogen bonding partner of the water molecule [27]. This active site configuration mimics a conformation that is presumably also present in reduced state wt-R2lox, but in a too small proportion of molecules to be modeled with confidence, and is assumed to be the conformation that is competent for O 2 binding and reduction, since with the fatty acid bound to the metal ions there are no free coordination sites for O 2 [24].…”

“…The external fatty acid ligand provides another bidentate bridge, and a water molecule is bound at the open coordination site of the Mn ion in site 1. Upon oxidation, E202 shifts outwards, leaving only the monodentate coordination to Fe2, while a hydroxo ion takes its place as a bridging ligand, and an ether cross-link is formed between the Cβ of V72 and the hydroxyl oxygen of Y162 close to the active site [23, 24, 26, 27]. Cross-link formation has been proposed to be initiated from the Mn IV /Fe IV state of the cofactor formed by oxygen reduction by abstraction of an electron from the tyrosine, with the radical subsequently transferred to the valine [23].…”

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

“…Mn1 retains a free coordination site, although this may be occupied by an additional water molecule that is not observed in the electron density (see XAS characterization below). The second-sphere residue Y175 is disordered, but best modelled in two alternate conformations: its usual conformation as a hydrogen bonding partner to the terminal water ligand of the site 1 metal ion, and a rotated conformation in which it may form a hydrogen bond with the non-binding carboxyl oxygen of E69 instead, otherwise the other hydrogen bonding partner of the water molecule (37). Y175 is somewhat disordered in reduced state wt-R2lox as well and may always be present in both conformations, just to different degrees (23,24).…”

“…Subsequent one-or two-electron/proton transfer events lead to the final, resting state cofactor structure. In the case of R2c the Mn IV and Fe III ions are linked by two oxo (µ-oxo/µ-hydroxo) bridges with a terminal hydroxo ligand on the Mn ion, whereas in R2lox the two metal(III) ions are linked by a single oxygen (µ-hydroxo) bridge, with the fatty acid ligand occupying the position of one of the oxygen bridges of R2c, and a terminal water ligand on the Mn ion ( Figure 1A, B) (16,23,(35)(36)(37). In R2lox, concomitant with cofactor activation, an ether cross-link is formed between the Cb of a valine and the hydroxyl oxygen of a tyrosine close to the active site.…”