Flavohemoglobin Detoxifies Nitric Oxide in Aerobic, but Not Anaerobic, Escherichia coli

Abstract

Nitric-oxide dioxygenase (NOD) and reductase (NOR) activities of flavohemoglobin (flavoHb) have been suggested as mechanisms for NO metabolism and detoxification in a variety of microbes. Mechanisms of NO detoxification were tested in Escherichia coli using flavoHb-deficient mutants and overexpressors. flavoHb showed negligible anaerobic NOR activity and afforded no protection to the NO-sensitive aconitase or the growth of anoxic E. coli, whereas the NOD activity and the protection afforded with O 2 were substantial. A NOinducible, O 2 -sensitive, and cyanide-resistant NOR activity efficiently metabolized NO and protected anaerobic cells from NO toxicity independent of the NOR activity of flavoHb. flavoHb possesses nitrosoglutathione and nitrite reductase activities that may account for the protection it affords against these agents. NO detoxification by flavoHb occurs most effectively via O 2 -dependent NO dioxygenation.Nitric oxide (NO) is a water-soluble gas commonly produced during the combustion of nitrogenous compounds and during the biological decay of organic matter. NO is also an important by-product of microbial denitrification (1, 2) and is produced by NO synthases of animals and plants, where it functions as a broad-spectrum antibiotic and signaling molecule (2-5). Nanomolar NO concentrations inactivate or inhibit critical enzymes, including the citric acid cycle enzyme aconitase (6 -8) and terminal respiratory oxidases (9,10). NO also has the potential to damage a variety of biomolecules by forming the more indiscriminate toxin and oxidizing agent peroxynitrite (11).Organisms have evolved mechanisms for NO metabolism and detoxification. Inducible nitric-oxide reductases (NORs) 1 are produced by denitrifying bacteria, nitrogen-dissimilating fungi, and pathogenic microorganisms. NORs reduce NO to N 2 O and are essential for denitrification by various bacteria and for the viability of gonococci (1,12,13). N 2 O is further reduced to N 2 by [4Cu-S]-containing N 2 O reductases in denitrifying microorganisms to generate energy through membrane-linked processes analogous to those utilized for O 2 respiration (1,14). In addition, NO-inducible flavohemoglobins (flavoHbs) dioxygenate NO to form NO 3 Ϫ (10, 15-21) and reduce NO to form N 2 O (19, 22).Growing evidence supports both aerobic and anaerobic NO detoxification functions for flavoHb. flavoHb protects aerobic Salmonella typhimurium against the growth inhibitory effects of acidified nitrite, S-nitrosoglutathione (GSNO), and the NOreleasing nitroso compound spermine 2,2Ј-(hydroxynitrosohydrazono)bisethanamine, and flavoHb prevents anaerobic growth inhibition of S. typhimurium by GSNO (23,24). flavoHb also protects Saccharomyces cerevisiae from aerobic and anaerobic growth inhibition by 2,2Ј-(hydroxynitrosohydrazono)bisethanamine (20). Pure NO gas (15) and nitroso compounds (19,25) potently inhibit the growth of Escherichia coli flavoHbdeficient mutants under aerobic growth conditions in contrast to flavoHb-containing parent strains, where growth inhibiti...

Nitric Oxide Homeostasis in Salmonella typhimurium

“…We have previously suggested (11) that NO accumulation in the hmp mutant inactivates FNR (18) with loss of up-regulation of the nitrite reductase responsible for NO generation. The present finding that mutation of hmp is without effect on anaerobic NO generation is consistent with the view that Hmp has primarily an O 2 -dependent NO-detoxifying role (51). Under aerobic conditions, however (Fig.…”

Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner

“…NO oxidation to nitrite and nitrate is also facilitated by transition metals and intracellular enzymes, e.g. by flavohemoglobin and truncated hemoglobin (25)(26)(27)(28)(29). Thus, the level of nitrite and nitrate in the medium indirectly reflects NOS activity.…”

Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species

“…For example, it has been proposed that homocysteine reacts with nitrosylating compounds and acts as an NO antagonist because S. enterica strains carrying insertions in metL encoding aspartokinase IIhomoserine dehydrogenase II are hypersensitive to Snitrosothiol (7). The most prominent detoxifying entities that have been identified are the NO dioxygenase, NO denitrosylase, and NO reductase activities associated with the hmpA-encoded flavohemoglobin and the NO reductase activity associated with the norVW-encoded flavorubedoxin and flavorubedoxin reductase (8)(9)(10)(11)(12)(13)(14)(15)(16). Other detoxifying activities include NO reductase activity contributed by nfrA-encoded periplasmic cytochrome c nitrite reductase (17), and GSNO reductase activity associated with the adhC-encoded alcohol-acetaldehyde dehydrogenase (18).…”