Investigation of Cyanide Ligand as an Active Site Probe of Human Heme Oxygenase.

Human heme oxygenase (hHO-1) is a physiologically important enzyme responsible for free heme catabolism. The enzyme's high regiospecificity is controlled by the distal site hydrogen bond network that involves water molecules and the D140 amino acid residue. In this work, we probe the active site environment of the wild-type (WT) hHO-1 and its D140 mutants using resonance Raman (rR) spectroscopy. Cyanide ligands are more stable than dioxygen adducts and are an effective probe of active site environment of heme proteins. The inherently linear geometry of the Fe-C-N fragment can be altered by the steric, electrostatic, and H-bonding interactions imposed by the amino acid residues present in the heme distal site, resulting in a tilted or bent configuration. The WT hHO-1 and its D140A, D140N, and D140E mutants were studied in the presence of natural abundance CN- and its isotopic analogues (13CN-, C15N-, and 13C15N-). Deconvolution of spectral data revealed that the ν(Fe-CN) stretching and δ(Fe-CN) bending modes are present at 454 and 376 cm-1, respectively. The rR spectral patterns of the CN- adducts of WT revealed that the Fe-C-N fragment adopts a tilted conformation, with a larger bending contribution for the D140A, D140N, and D140E mutants. These studies suggest that the FeCN fragment in hHO-1 is tilted more strongly toward the porphyrin macrocycle compared to other histidine-ligated proteins, reflecting the propensity of the exogenous hHO-l ligands to position toward the α-meso-carbon, which is crucial for the HO reactivity and essential for regioselectivity.