Desulfovibrio gigas Flavodiiron Protein Affords Protection against Nitrosative Stress In Vivo

Abstract

Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O 2 . Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas ⌬roo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O 2 reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.Desulfovibrio gigas is a sulfate-reducing bacterium considered to be a strict anaerobe. However, there is growing evidence that this organism can survive and probably even profit energetically from transient exposure to oxygen (8,23). Like other isolates from the Desulfovibrio genus, D. gigas has metalbinding enzymes for the detoxification of molecular oxygen (rubredoxin:oxygen oxidoreductase [ROO] and cytochrome bd) (8, 23), its reactive species (catalase and superoxide dismutase) (9), and superoxide reductase (34). The first proposed D. gigas defense mechanism against O 2 involved a flavometalloprotein, ROO (8), which accomplishes full oxygen reduction to water by accepting electrons from reduced rubredoxin (Rd). Rd is in turn reduced by NADH:rubredoxin oxidoreductase (7,8).ROO belongs to the flavodiiron protein (FDP) family, whose members are widespread in strict and facultative anaerobic Archaea and Bacteria members, as well as in anaerobic protozoa (1, 29, 30). The resolution of its crystallographic structure (12) revealed that each monomer of this homodimeric protein consists of two structural modules, a C-terminal flavin mononucleotide-binding flavodoxin-like domain and an N-terminal metallo-␤-lactamase-like domain. More recently, the structure of the FDP from Moorella thermoacetica was also solved (33).Although a role in oxygen reduction was initially assigned to the FDP family, recent reports contributed...

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

“…While the results here support the previously proposed idea of the role of ROO as a NO detoxifier (27,36), a more complex mechanism may explain the protection conferred by HCP. So far, HCPs have been described as having peroxidase activity (E. coli and D. desulfuricans ATCC 27774) (18) and hydroxylamine reductase activity (E. coli, Pyrococcus furiosus, and Rhodobacter capsulatus E1F1) (21)(22)(23).…”

“…In agreement, the D. gigas ROO was previously shown to be able to rescue an E. coli strain deleted in the NO-reductase flavodiiron gene and to have significant in vitro NO reductase activity (36).…”

Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors

“…In Desulfovibrio gigas, an equivalent enzyme complex was initially characterized as an oxidase, comprising the flavo-diiron protein (ROO, for rubredoxin oxygen oxido-reductase), a standalone rubredoxin and an NADH-rubredoxin oxidoreductase. The recombinant D. gigas ROO and reduced rubredoxin were found also to reduce NO to N 2 O, and physiological data suggested that the enzyme functions as an NO reductase in vivo [17]. Similarly, the D. vulgaris FprA functions as an NO reductase both in vitro and in vivo [18].…”

Flavodiiron Oxygen Reductase from Entamoeba histolytica

“…the FDPs from Moorella thermoacetica (8) and Desulfovibrio species (9,29)). The FDP from Escherichia coli, named flavorubredoxin (FlRd or NorV) (7,30), is selective toward NO (30,31).…”