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. 1998 Nov 24;95(24):14100-5.
doi: 10.1073/pnas.95.24.14100.

Nitrosative stress: metabolic pathway involving the flavohemoglobin

A Hausladen  1 , A J Gow, J S Stamler
Affiliations

Nitrosative stress: metabolic pathway involving the flavohemoglobin

A Hausladen et al. Proc Natl Acad Sci U S A. .

Abstract

Nitric oxide (NO) biology has focused on the tightly regulated enzymatic mechanism that transforms L-arginine into a family of molecules, which serve both signaling and defense functions. However, very little is known of the pathways that metabolize these molecules or turn off the signals. The paradigm is well exemplified in bacteria where S-nitrosothiols (SNO)-compounds identified with antimicrobial activities of NO synthase-elicit responses that mediate bacterial resistance by unknown mechanisms. Here we show that Escherichia coli possess both constitutive and inducible elements for SNO metabolism. Constitutive enzyme(s) cleave SNO to NO whereas bacterial hemoglobin, a widely distributed flavohemoglobin of poorly understood function, is central to the inducible response. Remarkably, the protein has evolved a novel heme-detoxification mechanism for NO. Specifically, the heme serves a dioxygenase function that produces mainly nitrate. These studies thus provide new insights into SNO and NO metabolism and identify enzymes with reactions that were thought to occur only by chemical means. Our results also emphasize that the reactions of SNO and NO with hemoglobins are evolutionary conserved, but have been adapted for cell-specific function.

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Figures

Figure 1
Figure 1
SNO and NO metabolism in E. coli. (A) E. coli release NO from S-nitrosothiols. S-nitrosocysteine (0.1 mM) was added to growth medium (broken line) or a suspension of E. coli (A 600 = 1; 2 × 109 cfu/ml) in growth medium (solid line), and NO release was measured with an NO electrode. (B) E. coli consume NO in both a constitutive and an inducible manner. A saturated solution of NO was added to growth medium (dotted line), to a suspension of E. coli that had received no treatment (dashed line), or to E. coli pretreated with 0.2 mM SNO-Cys for 90 min (solid line). (C) E. coli contain SNO-lyase activities. A bacterial extract was separated by anion-exchange chromatography, and the column fractions were assayed for the accumulation of nitrite from 0.5 mM SNO-Cys. (D) E. coli contain an inducible NO-metabolizing heme protein. Absorption spectra of column fractions from untreated (dashed line) or SNO-Cys pretreated cells (solid line), which contain the inducible NADH-dependent NO-metabolizing activity.
Figure 2
Figure 2
HMP is required for NO consumption and resistance to nitrosative stress. (A) NO metabolism. NADH-dependent consumption of 10 μM NO by extracts (1 mg/ml protein) from untreated Δhmp cells (dotted line, top), SNO-Cys-pretreated Δhmp cells (long-dashed line), untreated wild-type cells (short-dashed line), and SNO-Cys-pretreated wild-type cells (solid line). (B) Cytostatic effect of SNO-Cys treatment on isogenic wild-type (wt) and Δhmp mutant strains (hmp). +/+ and −/−, Cells that had or had not been pretreated with 0.2 mM SNO-Cys for 90 min, respectively, and were then rechallenged with the same dose. −/−, Control (wt or Δhmp mutant) cells that were neither pretreated nor rechallenged with SNO-Cys. In both strains, A600 is proportional to cell mass (protein content) at baseline and after treatment. Pretreatments were initiated at A600 = 0.2 for 90 min.
Figure 3
Figure 3
NO oxygenase activities of purified HMP. (A) NO consumption. NO electrode signal after addition of ≈10 μM NO to buffer (solid line) or ≈10 μM NO (dotted line) or ≈35 μM NO (dashed line) to 40 μg/ml HMP + 0.1 mM NADH. (B) Absorption spectra of purified HMP. HMP (450 μg/ml) exhibited an oxidized spectrum under anaerobic conditions (line 1, dashed–dotted); addition of NADH produced a ferrous iron-like spectrum (line 2, dashed); addition of NO saturated solution generated an iron-nitrosyl spectrum (line 3, solid); and air exposure of this iron-nitrosyl (with brief vortexing) resulted in an oxygen-bound (ferrous) iron spectrum (line 4, dotted). (C) Absorption spectra of HMP during aerobic turnover of NO in the presence of NADH. NADH (300 μM) was added to 400 μg/ml HMP in air (dotted line); addition of 100 μM NO from a saturated solution resulted in the consumption of NADH but no loss of the oxygen-bound ferrous iron spectrum (solid line). (D) Aerobic NADH consumption is increased during NO turnover by HMP and is inhibited by cyanide. NO (100 μM) was generated from SNO-Cys/Cu2+ (added to 20 μg/ml HMP at the break) in the absence (solid line) or presence (dotted line) of 1 mM KCN. (E) Product formation by HMP is not significantly influenced by SOD. Nitrite (solid bars) and nitrate (open bars) yields after addition of DEANO to 20 μg/ml HMP in the presence of 0.1 mM NADH and the indicated amounts of SOD. (F) NO oxidation by HMP (solid line) increases oxygen-consumption rate over NO autooxidation (broken line); the stoichiometry is 1 NO per O2 consumed. Where indicated by the asterisks (∗), 100 μM NO was added.
Figure 4
Figure 4
Metabolic pathways for SNO and NO. SNO is cleaved to NO by constitutive SNO-lyases. In aerobic metabolism NO is oxidized to nitrate and nitrite (NOx) by the HMP-dioxygenase, the levels of which are up-regulated by nitrosative stress. The accumulation of nitrite is prevented by OxyR-controlled genes (6), although the mechanism is unknown. Under anaerobic conditions, NO/SNO are at least partly metabolized to N2O (not shown).
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