Interaction of Nitric Oxide with Human Heme Oxygenase-1

Abstract

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 Fe 3؉ -NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide⅐sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The (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 Fe 3؉ -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 Fe 3؉ -NO complex is ϳ50-times faster, and k off 10 times slower, than for met-Mb, resulting in K d ‫؍‬ 1.4 M 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. Nitric oxide (NO)1 functions as a signaling molecule in a diversity of physiological responses, including vasodilation and regulation of normal vascular tone, neuronal signal transmission, cytotoxicity against pathogens and tumors, and regulation of cellular respiration (1-3). Most of these responses result from interaction of NO with the heme group of the receptor guanylyl cyclase (1). A role akin to that of NO in signaling pathways has also been postulated for CO (4). CO is produced in mammals from heme by two heme oxygenases, HO-1 and HO-2 (5-8). The involvement of CO has been invoked as a factor in atherosclerosis (9), psoriasis (10), vascular constriction (11), chronic renal inflammation (12), cellular protection (13), hyperoxia-induced lung injury (14), and other physiological situations. The role of CO as an NO-like signaling molecule has received strong support from studies of heme oxygenase and nitric-oxide synthase knockouts (15), but much of the evidence, particularly that which depends heavily on inhibition of heme oxygenase by metalloporphyrins such as tin protoporphyrin IX, is tainted by ambiguities concerning the specificity of the inhibitors (16). Nevertheless, the collective evidence makes a persuasive case for at least a limited role for CO in mammalian signaling systems.Evidence has accumulated that interactions of CO and NO may influence the physiological responses to each of these agents thr...

Expression and Characterization of Full-Length Human Heme Oxygenase-1: The Presence of Intact Membrane-Binding Region Leads to Increased Binding Affinity for NADPH Cytochrome P450 Reductase

“…Some studies have been done using fulllength HO-1 from other species (8,9,(16)(17)(18); but most studies, including recent crystal structures and catalytic characterizations of human HO-1 have focused on the truncated forms (12,21,(31)(32)(33)(34)(35)(36)(37)(38). A 30-kDa soluble hHO-1, which lacks the 23 C-terminal amino acids that are thought to serve as the membrane spanning region of the enzyme, was purified and crystallized (12,21).…”

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

“…50 Porphyrin skeletal vibrations observed in the high-frequency range of RR spectra, obtained with Soret excitations support a low-spin description of {FeNO} 6 complexes. 30,[50][51][52][53][54] The ν 3 and ν 10 porphyrin core modes are observed at higher frequencies in {FeNO} 6 than in 6-coordinate {FeNO} 7 species. 30,[50][51][52][53][54] Importantly, vibrational modes of the FeNO unit in {FeNO} 6 species do not seem to correlate with those of {FeNO} 7 and carbonyl complexes.…”

“…[24][25][26] Ferrous hemoproteins typically exhibit a very high affinity for NO while the ferric forms bind NO with K d values in the micromolar range. [27][28][29][30] Because the concentration of NO during denitrification remains well below the micromolar range, it is assumed that the catalytic cycle of NO reductases is initiated by the binding of NO to an iron(II) in the reduced form of the enzyme.…”

Potential role of heme oxygenase-1 in the progression of rat adjuvant arthritis

“…For instance, CO may elevate the steady-state level of NO leading to peroxynitrite production by endothelial cells and perivascular oxidative injury, 19 whereas NO can bind to human HO-1 leading to reversible inhibition of this enzyme. 20 In fact, endogenous NO functions as an inhibitory regulator of CO production by HO-1 in rat kidney. 21 In addition, a synergy between NO and CO to provide cytoprotection has been reported in tumor necrosis factor-a (TNFa)-induced hepatocyte cell death.…”