Steady-state and Transient Kinetics of Escherichia coli Nitric-oxide Dioxygenase (Flavohemoglobin)

Abstract

The flavoHbs 1 belong to a 1.8 billion-year-old family of globin molecules that includes O 2 -binding Hbs and Mbs isolated from animals, plants, fungi, protozoa, bacteria, and worms (1-6). FlavoHbs have a unique two-domain structure containing linked Hb and reductase domains with extensive homology to the mammalian Hbs and metHb reductases (1, 7). Other Hbs appear to be co-expressed with associated metHb reductases (8). An O 2 transport or storage function, like that of the erythrocytic Hbs and muscle Mbs, has been suggested for some microbial and plant (flavo)Hbs (4, 9); however, other functions including the catalysis of oxidations have long seemed more likely (10 -12).Recently, we described an NO dioxygenase (NOD) produced by Escherichia coli that utilizes O 2 and NAD(P)H to convert NO to nitrate (Equation 1) and identified it as a flavoHb (13,14). Subsequent studies have shown that related bacterial and yeast flavoHbs display a similar NOD activity.A role for flavoHbs in NO detoxification is supported by the ability of flavoHbs to protect bacteria against NO or nitroso compounds (13, 14, 16 -18) and by their induction in bacteria exposed to NO, nitrate, nitrite, or nitroso compounds (13, 14, 17, 19 -22). However, the mechanism of NO detoxification, and thus the function of the flavoHbs, is obscured by the possibility of multiple reaction mechanisms involving NO. Other NO detoxifying activities for flavoHbs, including denitrosylation of nitrosothiols (17), NO reduction (17, 23), and NO sequestration (16, 23), have been offered to explain the protection flavoHbs afford to bacteria against NO and nitrosoglutathione. Thus, an understanding of the biological function(s) of the flavoHbs demands a greater knowledge of their various activities.In this report, steady-state, reduction, and ligand binding kinetics of the E. coli NOD (flavoHb) were measured in order to define its function and the mechanism of NO dioxygenation. We also examined the effects of amino acid substitutions at the highly conserved Tyr(B10) position on NOD activity, reduction, and ligand binding kinetics (7,24). Key differences between flavoHb and other Hbs are discussed in light of this specialized but perhaps ancient NO dioxygenation and detoxification function of hemoglobin. MATERIALS AND METHODSCells and Reagents-The flavoHb-deficient E. coli strain RB9060 (25) was generously provided by Alex Ninfa (University of Michigan). Plasmid pAlter containing the E. coli hmp gene was prepared as described previously (13). FAD, NADPH, and bovine hemin were purchased from Sigma. NADH, bovine liver catalase (260,000 units/ml), and deoxyribonuclease were obtained from Roche Molecular Biochemicals. Saturated NO was prepared as described previously (26). Saturated O 2 (1.14 mM) was prepared by vigorously scrubbing a solution of 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA (buffer A) at 25°C and atmospheric pressure with 99.993% O 2 (Praxair, Bethlehem, PA) in a rubber septum-sealed glass vial vented with a syringe needle. Manganese-containing...

A Conserved Val to Ile Switch near the Heme Pocket of Animal and Bacterial Nitric-oxide Synthases Helps Determine Their Distinct Catalytic Profiles

“…The Fe III NO dissociation rates of mammalian NOS enzymes are within a range that is typical for other ferric heme proteins, whereas in bsNOS this rate is near the lower end of the range (Table V). In contrast, the rates of ferric heme reduction in mammalian NOS enzymes range between 0.1 and 4 s Ϫ1 at 10°C, which are much slower than in other heme enzymes that contain attached flavin domains like cytochrome P450BM3 (23) or flavohemoglobin (24). We suspect that this circumstance evolved in the mammalian NOS to enable their NO release from the heme.…”

Nonsymbiotic hemoglobins in rice are synthesized during germination and in differentiating cell types

“…In addition, given the in vivo localization of ns-Hbs in metabolically active tissues and the very high affi nity for oxygen binding of several r-ns-Hbs, it is unlikely that they function as oxygen scavengers (Arredondo-Peter et al 1998, Hill 1998). Both Arredondo-Peter et al (1998) and Hill (1998) have suggested that plant ns-Hbs could act as NO scavengers, in a similar manner to those of the bacterial fl avohemoglobins, which have been documented to possess nitric oxide dioxygenase properties (R Gardner et al 1998, A. Gardner 2000. However, nsHbs lack a fl avin domain, which would be required for functioning as an NO dioxygenase.…”

Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coli – Implications for urinary tract infection

“…The decreased viability of the J96∆norV mutant may indicate that the oxygen level in the static culture in our experiments is microaerobic rather than aerobic. In hypoxic conditions the dioxygenase activity of Hmp have been shown to be reduced [42], which may explain why Hmp was unable to compensate for the norV-deficiency. Taken together, our results suggest that, under the experimental conditions used, both Hmp and NorV seem to be important for NO protection in UPEC.…”