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The Protonation Status of Compound II in Myoglobin, Studied by a Combination of Experimental Data and Quantum Chemical Calculations: Quantum Refinement

Kristina Nilsson ^*, Hans-Petter Hersleth , Thomas H. Rod ^*, K. Kristoffer Andersson  and Ulf Ryde ^*

^* Department of Theoretical Chemistry, Lund University, Lund, Sweden; and Department of Chemistry and Department of Molecular Bioscience, University of Oslo, Blindern, Oslo, Norway

Correspondence: Address reprint requests to U. Ryde, Tel.: 46-46-2224502; Fax: 46-46-2224543; E-mail: ulf.ryde{at}teokem.lu.se.

Treatment of met-myoglobin (Fe^III) with H₂O₂ gives rise to ferryl myoglobin, which is closely related to compound II in peroxidases. Experimental studies have given conflicting results for this species. In particular, crystallographic and extended x-ray absorption fine-structure data have shown either a short (170 pm) or a longer (190 pm) Fe–O bond, indicating either a double or a single bond. We here present a combined experimental and theoretical investigation of this species. In particular, we use quantum refinement to re-refine a crystal structure with a long bond, using 12 possible states of the active site. The states differ in the formal oxidation state of the iron ion and in the protonation of the oxygen ligand (O^2–, OH^–, or H₂O) and the distal histidine residue (with a proton on N¹, N², or on both atoms). Quantum refinement is essentially standard crystallographic refinement, where the molecular-mechanics potential, normally used to supplement the experimental data, is replaced by a quantum chemical calculation. Thereby, we obtain an accurate description of the active site in all the different protonation and oxidation states, and we can determine which of the 12 structures fit the experimental data best by comparing the crystallographic R-factors, electron-density maps, strain energies, and deviation from the ideal structure. The results indicate that Fe^III OH^– and Fe^IV OH^– fit the experimental data almost equally well. These two states are appreciably better than the standard model of compound II, Fe^IV O^2–. Combined with the available spectroscopic data, this indicates that compound II in myoglobin is protonated and is best described as Fe^IV OH^–. It accepts a hydrogen bond from the distal His, which may be protonated at low pH.