The protonation status of compound II in myoglobin, studied by a combination of experimental data and quantum chemical calculations: quantum refinement

Abstract

Treatment of met-myoglobin (FeIII) 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 (approximately 170 pm) or a longer (approximately 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 (O2-, OH-, or H2O) and the distal histidine residue (with a proton on Ndelta1, Nepsilon2, 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 FeIII OH- and FeIV OH- fit the experimental data almost equally well. These two states are appreciably better than the standard model of compound II, FeIV O2-. Combined with the available spectroscopic data, this indicates that compound II in myoglobin is protonated and is best described as FeIV OH-. It accepts a hydrogen bond from the distal His, which may be protonated at low pH.

FIGURE 1

The proposed reaction cycle for peroxidases (Gajhede, 2001). Compounds I and II are complexes 5 and 6.

FIGURE 2

The small Fe(Por)(Im)(OH⁻) model of the heme active site used in some of the vacuum calculations.

FIGURE 3

The Fe(Por)(Im)(OH⁻)(Im) model of the active heme site used in both the vacuum and quantum-refinement calculations. The three figures illustrate the three possible protonation states of the distal His model—HID, HIE, and HIP.

FIGURE 4

A comparison of the original crystal structure (magenta) and the re-refined structure of Fe^IV OH⁻ HIE. Also included is the f_o−f_c difference map of the original crystal structure at the ±3.0σ level (cyan and yellow).

FIGURE 5

A comparison of the ComQum-X structures of the Fe^III OH⁻ HIE (magenta) and Fe^IV OH⁻ HIE models. Also included are the f_o−f_c difference maps of the two structures at the ±3.0σ level (blue and white for Fe^III and green and red for Fe^IV).