Escherichia coli Hmp, an ?oxygen-binding flavohaemoprotein?, produces superoxide anion and self-destructs

Abstract

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k(on) 2.82x10(7) M(-1) s(-1) and k(off) 4.49x10(3) s(-1). The dissociation constant of the primary O(2) compound was 160 microM (25 degrees C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K(m) <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.

Mechanisms of Adaptation to Nitrosative Stress inBacillus subtilis

“…It has been shown that E. coli Hmp is irreversibly inactivated in the presence of O 2 and NADH due to superoxide formed during turnover. However, in the presence of NO, superoxide production is abolished (40). Thus, we speculate that a small amount of NO induces hmp expression and protects the flavohemoglobin from self-destruction.…”

Binding of Azole Antibiotics to Staphylococcus aureus Flavohemoglobin Increases Intracellular Oxidative Stress

“…This hypothesis is supported by the ability of several flavin-containing proteins to generate superoxide upon oneelectron oxidation by dioxygen (10). Also, it was previously shown that NAD(P)H is oxidized by flavohemoglobin with the electrons transferred via FAD to external acceptors when the ferrous heme is blocked (28).…”

“…It has been reported that, under certain conditions, E. coli flavohemoglobin produces superoxide ions (16,28). To investigate the effect of antibiotic binding to flavohemoglobin on radical formation, we used the spin trap BMPO in EPR experiments.…”

Peroxynitrite stress is exacerbated by flavohaemoglobin-derived oxidative stress in Salmonella Typhimurium and is relieved by nitric oxide

“…Under aerobic conditions and in the absence of NO, Hmp forms the highly reactive species O { : 2 via the reduction of molecular oxygen (Membrillo-Hernández et al, 1996;Orii et al, 1992;Wu et al, 2004); however, in the presence of NO, the relatively inert nitrate ion is formed in a denitrosylase or dioxygenase reaction (Gardner et al, 2000;Hausladen et al, 2001;Poole & Hughes, 2000). As Hmp levels are elevated in an nsrR mutant (Fig.…”

“…Aerobically, Hmp catalyses the reaction between oxygen and NO to produce relatively inert nitrate (Gardner et al, 1998;Hausladen et al, 1998). In the absence of NO, O { : 2 is formed, which reacts with a variety of compounds leading to the formation of other oxidative stresses such as hydrogen peroxide (H 2 O 2 ) (MembrilloHernández et al, 1996;Orii et al, 1992;Wu et al, 2004). Anaerobically, NO reacts with the haem of Hmp to yield a nitrosyl anion with subsequent formation of nitrous oxide (Hausladen et al, 1998;Kim et al, 1999;Liu et al, 2000).…”