Solution 1H NMR of the Active Site of Substrate-bound, Cyanide-inhibited Human Heme Oxygenase: COMPARISON TO THE CRYSTAL STRUCTURE OF THE WATER-LIGATED FORM

Gerd N La Mar,Anbanandam Asokan,Bryan Espiritu,Deok Cheon Yeh,Karine Auclair,Paul R Ortiz De Montellano

doi:10.1074/jbc.m009974200

Abstract

The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer ( approximately 75-80%) in solution is rotated 180 degrees about the alpha-gamma-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 A closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a approximately 20 degrees tilt of the distal ligand from the heme normal in the direction of the alpha-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta, gamma, and delta-meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha-meso position.

Highlights

The majority of the active site residues of cyanideinhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide
Heme oxygenase (HO)[1] is a membrane-bound protein that carries out the NADPH, O2, and cytochrome P450 reductasedependent regiospecific catabolism of heme to iron, ␣-biliverdin, and carbon monoxide (1)
Previous assignments by isotope labeling in rat HO-hemin-CN (16) and twodimensional NMR on human HO (hHO)-hemin-CN (18) had assigned M3 to the 3-CH3 peak of the complex with the heme in the orientation shown in Fig. 1A and m3 to the 3-CH3 of the complex with the heme rotated by 180° about the ␣-␥-meso axis

Summary

Introduction

The majority of the active site residues of cyanideinhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide His-25 and Glu-29 C␤Hs. Lastly, the labile proton of the TOCSY-detected CH3CH-OH fragment exhibits a NOESY cross-peak to 8-CH3 (Fig. 3E), as expected for the Thr-21 to 5-CH3 contact in the crystal structure (24).

Results

Conclusion