Interaction of nitric oxide with human heme oxygenase-1

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe³⁺-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethyl-amino)-diazenolate-2-oxide·sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The v(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm^-1 lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe³⁺-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k_on for formation of the hHO-1-heme Fe³⁺-NO complex is ∼50-times faster, and k_off 10 times slower, than for met-Mb, resulting in K_d = 1.4 μM for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K_d value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.