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. 1998 Sep 1;95(18):10378-83.
doi: 10.1073/pnas.95.18.10378.

Nitric oxide dioxygenase: an enzymic function for flavohemoglobin

P R Gardner  1 , A M Gardner, L A Martin, A L Salzman
Affiliations

Nitric oxide dioxygenase: an enzymic function for flavohemoglobin

P R Gardner et al. Proc Natl Acad Sci U S A. .

Abstract

Nitric oxide (NO*) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO*-resistant mutants of Escherichia coli were isolated that rapidly consumed NO*. An NO*-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3-. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E. coli flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO*. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO*. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO* detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage.

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Figures

Figure 1
Figure 1
Elevated NO• consumption by NO•-resistant mutants of E. coli. (A) Cultures were grown in phosphate-buffered LB medium with minimal aeration (open bars), with vigorous shaking under air (hatched bars), or under an atmosphere containing 960 ppm NO• in 21% O2 balanced with N2 for 60 min (filled bars), and cells were harvested and washed and NO• consumption was measured. (B) A total of 2.5 million cells from logarithmic cultures of AB1157, PG110, and PG118 were plated on phosphate-buffered LB agar medium containing phanazine methosulfate (50 μM) and were exposed to 960 ppm NO• under an atmosphere of 10% air balanced with N2 or to air at 37°C for 16 h. Cultures were then incubated at 37°C under a normal air atmosphere for 24 h to allow the outgrowth of bacterial colonies. Colonies were counted and percent survival was calculated relative to air exposed controls (n = 2; ±SD).
Figure 2
Figure 2
Formation of NO3− by mutant extracts. NO3− formation in NO• consumption reactions catalyzed by 150 μg of PG118 (line 1) or AB1157 (line 2) extract protein was measured in a 10 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.2 mM NADP+, 2.5 mM glucose-6-phosphate, 0.5 unit/ml glucose-6-phosphate dehydrogenase, 1 μM FAD, and 10 μg/ml bovine erythrocyte copper and zinc-containing superoxide dismutase. Superoxide dismutase was included to inhibit NO2− and NO3− formation by the O2⨪-dependent oxidation of NO•. The reaction was equilibrated with an atmosphere containing 480 ppm NO• in 10% air balanced with N2 by vigorous shaking in a gyrorotatory water bath at 37°C. NO2− formation with PG118 (line 3) and AB1157 (line 4) extracts was also assayed. Extract-catalyzed NO3− and NO2− formation were calculated by subtracting the amount of extract-independent NO3− and NO2− formation. The amount of PG118 and AB1157 extract added was sufficient to catalyze the conversion of 47 and 0.3 μM NO• per min in the standard NO• consumption assay, respectively. Error bars represent SD where n = 3.
Figure 3
Figure 3
Identification of a unique FAD-binding protein in cell-free extracts of the NO•-resistant mutant PG118. Cell-free extracts at 25 μg (lanes 1 and 2), dialyzed 33–55% ammonium sulfate fractions at 25 μg (lanes 3 and 4), and proteins eluted from FAD-agarose (lanes 5 and 6) were separated on 8–16% SDS/PAGE and stained with Coomassie blue. Samples from AB1157 were separated in lanes 1, 3, and 5, and the corresponding PG118 samples were in lanes 2, 4, and 6. FAD-agarose (0.1 ml) was boiled in SDS sample buffer following an overnight incubation with an equal volume of buffer A containing 2 mg protein from the ammonium sulfate fractions and following extensive washing. One-twentieth of the total amount eluted was loaded on the gel.
Figure 4
Figure 4
Fractionation of NOD from a mutant overexpressor. (A) Fractions were separated on a 8–16% SDS/PAGE gel and stained with Coomassie blue. Lanes: 1, cell-free lysate, 25 μg; 2, 33–55% ammonium sulfate dialysate, 25 μg; 3, FAD-agarose eluate concentrate, 2 μg; 4, Superdex 200 pooled concentrate, 1.2 μg; and 5, Mono Q 0.2 M NaCl eluate concentrate, 2.9 μg. (B) Superdex 200 elution profile of the NOD activity and protein. The arrow points to the elution point of the marker BSA.
Figure 5
Figure 5
Effects of flavohemoglobin expression on cellular NO• consumption activity and NO• toxicity. (A) NO• consumption activities of the hmp deletion mutant (RB9060), its parent (YMC10), the flavohemoglobin overexpressor strain JM109 containing hmp in the multi-copy plasmid vector pAlter (+ hmp), and its control strain containing pAlter only (JM109) were measured during growth in air (open bars) or following a 60-min exposure to 960 ppm NO• balanced with N2 and 21% O2 (filled bars). Strains YMC10 (lines 1 and 2) and RB9060 (lines 3 and 4) were grown continuously under air (B) or N2 (C), or were exposed after 60 min (lines 2 and 4) to 960 ppm NO• balanced with N2 and 21% O2 (B) or to 240 ppm NO• balanced with N2 (C). Growth was monitored at 550 nm. Aerobic and anaerobic cultures were initiated in phosphate-buffered LB medium with 2% inocula from overnight aereobic cultures and with 4% inocula from static overnight anoxic cultures, respectively. Glucose (20 mM) was added for anaerobic growth. Cells were treated at an A550 = 0.3 for measurements of NO• consumption activity. Error bars represent the SD of three measurements.
Scheme 1
Scheme 1
.
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