Solution NMR study of environmental effects on substrate seating in human heme oxygenase: Influence of polypeptide truncation, substrate modification and axial ligand

Abstract

Solution proton NMR has been used here to show that, as either the high-spin ferric, protohemin (PH) substrate complex at neutral pH, or the low-spin ferric, cyanide-inhibited PH substrate complex, the active site electronic and molecular structure of the 233- and 265-residue recombinant constructs of human heme oxygenase-1, hHO, are essentially indistinguishable. It is shown, moreover, that the equilibrium PH orientational isomerism about the α,γ- meso axis is 1:1 in the water-ligated, resting-state complex, but changes to a 4:1 equilibrium ratio as the cyanide-inhibited complex, with the minor species in solution corresponding to the only one found in crystals. The introduction of significant PH orientational preference in the cyanide over the aquo complex is rationalized by the crystallographic observation for the same H 2O and CN ligated complexes of rat heme oxygenase (rHO), where the steric tilt of the Fe–CN unit resulted in a ∼1 Å transition of PH into the hydrophobic interior, and stronger interaction of the vinyls with the HO matrix [M. Sugishima, H. Sakamoto, M. Noguchi, K. Fukugama, Biochemistry 42 (2003) 9898–9905]. 1H NMR spectra of the cyanide-inhibited PH complex are the most used, and most useful, for determining the distribution of orientational isomerism for PH in complexes of HO. Hence, it is imperative that the time-course of the spectra after sample preparation be considered in order to reach conclusions that relate isomeric seating of the heme with variable isomeric biliverdin products. The natural orientational isomerism of PH leads to spectral congestion that has prompted the use of a synthetic, twofold symmetric substrate, 2,4-dimethyldeuterohemin, DMDH. While the hyperfine shift pattern for non-ligated residues are very similar and are consistent with largely conserved molecular structure with the alternate substrates, the steric tilt of the Fe–CN vector towards the protein interior, as determined by the orientation of the major magnetic axes, is 2° smaller for DMDH than PH, and is rationalized by the substrate translating even further into the hydrophobic interior in the cyanide complex when the bulky vinyl groups are replaced by methyl groups.