Nitric-oxide Dioxygenase Activity and Function of Flavohemoglobins

Abstract

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O 2 (K I (CO) ‫؍‬ ϳ1 M). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O 2 from inhibitory NO and CO.Investigations of NO toxicity and defenses in Escherichia coli led to the isolation of an NO-inducible NAD(P)H, O 2 , and FAD-dependent NOD 1 activity. This enzyme activity was identified with the flavoHb encoded by the hmp gene (1, 2). More recently, we have shown that the flavoHb efficiently converts NO and O 2 to nitrate via a conventional two electron flavoenzyme mechanism (3). The proposed NOD reaction stoichiometry is described by Equation 1.A function for flavoHbs in NO dioxygenation and detoxification is supported by a growing body of evidence. FlavoHbs are induced by NO, nitrite, nitrate, and NO-releasing agents in various bacteria (2, 4 -9), and these flavoHbs protect bacteria, yeast, and Dictyostelium discoideum against growth inhibition and NO-mediated damage during exposures to authentic NO or NO releasing agents (1, 2, 6, 7, 10 -12). Further, O 2 is required for the robust NO scavenging action of flavoHb both in E. coli and in vitro, and O 2 is required for the maximal protection of cells and NO-sensitive aconitases from NO-mediated damage (1, 2). Other flavoHb mechanisms may also protect cells against nitrosothiol or NO toxicity in the absence of O 2 . FlavoHbs may reduce NO or denitrosylate toxic nitrosothiols formed from NO, sequester NO or reactive heme, or catalyze other beneficial reactions (6,10,13,14). These additional flavoHb activities and functions require consideration.We have now investigated the NOD activities of flavoHbs isolated from Saccharomyces cerevisiae, Alcaligenes eutrophus, and E. coli and have compared these activities with other enzymic activities of flavoHbs including NO reductase, NADH oxidase, and FAD reductase activities. Spectra for the various O 2 , NO, and CO liganded flavoHbs and the transient kinetics for O 2 , NO, and CO binding are reported for flavoHbs and are discussed in relation to the susceptibility of NOD activity to NO and CO inhibition. The kinetics and stoichiometry of the NOD activity indicate a highly specific and efficient dioxygenase mechanism and function for the flavoHbs. MATERIALS AND METHODSReagents-NADPH, FAD, phenylmethylsulfonyl fluoride, and Aspergillus niger glucose oxidase (225 units/mg) were purchased from Sigma. NADH, Aspergillus nitrate reductase, and bovine liver catalase

Heme-Ligand Tunneling in Group I Truncated Hemoglobins

“…TrHbs from more than one group can coexist in some bacteria, suggesting a wide diversification of functions. Possible trHb functions that are consistent with observed biophysical properties include long term ligand or substrate storage, NO detoxification, O 2 /NO sensor, redox reactions, and O 2 delivery under hypoxic conditions (1)(2)(3). In Mycobacterium bovis BCG, trHbN promotes an efficient dioxygenase reaction whereby NO is converted to nitrate by the oxygenated heme (4).…”

“…1). In contrast, the recently solved crystal structure of hexacoordinated Ss-trHb, where the sixth heme ligand is residue HisE10, 2 did not show evidence of such cavity network, likely related to extensive conformational changes observed in the protein distal site region (8).…”

Human Disease-related Mutations in Cytochrome b Studied in Yeast

“…It is interesting to note that in the respiratory deficient mutants, as shown for ⌬252-259 in Fig. 2, the aerobic spectrum (dotted line) showed a peak at 562 nm, corresponding to reduced cytochrome b, presumably b h , and a small peak at 575 nm, corresponding to oxyflavohemoprotein (16). Upon addition of dithionite (solid line), cytochromes c, c 1 , and oxidase (aa 3 ) were reduced and the signal of oxyflavohemoprotein disappeared.…”

Isocitrate Dehydrogenase Is Important for Nitrosative Stress Resistance in Cryptococcus neoformans, but Oxidative Stress Resistance Is Not Dependent on Glucose-6-Phosphate Dehydrogenase

“…NADPH is used as an electron donor either in recycling of oxidized, inactive enzymes to reduced, active forms or directly in catalytic activity. For example, Fhb1 binds NADPH during its catalytic activity and uses it directly as an electron donor for the reduction of NO ⅐ to NO 3 (21). Catalases, which are highly conserved antioxidants that dismute H 2 O 2 to molecular oxygen and water, consist of four units each with a molecule of NADPH bound in the core (18,36,59).…”