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.
Enzymes with a dimetal-carboxylate cofactor catalyze reactions among the top challenges in chemistry such as methane and dioxygen (O2) 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 O2reduction 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 (H2O) 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-OH2bond 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 O2reduction and cross-link formation reactions are discussed.
Fachbereich Physik, Freie Universität Berlin , 14195 Berlin, Germany.
Ramona
R
Kositzki
Fachbereich Physik, Freie Universität Berlin , 14195 Berlin, Germany.
Stefan
S
Mebs
Fachbereich Physik, Technische Universität Kaiserslautern , 67663 Kaiserslautern, Germany.
Jennifer
J
Marx
Department of Biochemistry and Biophysics, Stockholm University , 10691 Stockholm, Sweden.
Julia J
JJ
Griese
Fachbereich Physik, Freie Universität Berlin , 14195 Berlin, Germany.
Nils
N
Schuth
Department of Biochemistry and Biophysics, Stockholm University , 10691 Stockholm, Sweden.|Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Martin
M
Högbom
Fachbereich Physik, Technische Universität Kaiserslautern , 67663 Kaiserslautern, Germany.
Volker
V
Schünemann
Fachbereich Physik, Freie Universität Berlin , 14195 Berlin, Germany.
Michael
M
Haumann