RESUMO
The flavohaemoglobin gene, hmp, of Escherichia coli is upregulated by nitric oxide (NO) in a SoxRS-independent manner. We now show that hmp expression is also upregulated by S-nitrosoglutathione (GSNO, widely used as an NO releaser) and sodium nitroprusside (SNP, which is a NO+ donor). Elevated homocysteine (Hcy) levels, achieved either by adding Hcy extracellularly or using metE mutants, decreased hmp expression. Conversely, metC mutants (defective in Hcy synthesis) had higher levels of hmp expression. Mutations in metR abolished hmp induction by GSNO and SNP, and hmp expression became insensitive to Hcy. We propose that the previously documented modulation by Hcy of MetR binding to the glyA-hmp intergenic regulatory region regulates hmp transcription. Although two MetR binding sites are present in this region, only the higher affinity site proximal to hmp is required for hmp induction by GSNO and SNP. GSNO and SNP react with Hcy in vitro under physiologically relevant conditions of pH and temperature generating S-nitrosohomocysteine, although in the latter case this would be co-ordinated to the Fe in SNP as a stable species. The free S-nitrosocysteine generated in the reaction with GSNO breaks down to release NO more readily than via homolysis of GSNO. As GSNO and SNP upregulate hmp similarly, the NO released in the former case on reaction with homocysteine cannot be involved in hmp regulation.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase , Proteínas de Bactérias/genética , Di-Hidropteridina Redutase , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Hemeproteínas/genética , NADH NADPH Oxirredutases , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glutationa/análogos & derivados , Glutationa/farmacologia , Glicina Hidroximetiltransferase/genética , Homocisteína/metabolismo , Metiltransferases , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , S-Nitrosoglutationa , Transativadores/genética , Transativadores/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Escherichia coli possesses a flavohemoglobin (Hmp), product of hmp, the first microbial globin gene to be sequenced and characterized at the molecular level. Although related proteins occur in numerous prokaryotes and eukaryotic microorganisms, the function(s) of these proteins have been elusive. Here we report construction of a defined hmp mutation and its use to probe Hmp function. As anticipated from up-regulation of hmp expression by nitric oxide (NO), S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP), the hmp mutant is hypersensitive to these agents. The hmp promoter is more sensitive to SNP and S-nitroso-N-penicillamine (SNAP) than is the soxS promoter, consistent with the role of Hmp in protection from reactive nitrogen species. Additional functions for Hmp are indicated by (a) parallel sensitivity of the hmp mutant to the redox-cycling agent, paraquat, (b) inability of the mutant to up-regulate fully the soxS and sodA promoters in response to oxidative stress caused by paraquat, GSNO and SNP, and (c) failure of the mutant to accumulate reduced paraquat radical after anoxic growth. We conclude that Hmp plays a role in protection from nitrosating agents and NO-related species and oxidative stress. This protective role probably involves direct detoxification of those species and sensing of NO-related and oxidative stress.