A NOVEL MECHANISM FOR UP-REGULATION OF THE ESCHERICHIA-COLI K-12 HMP (FLAVOHAEMOGLOBIN) GENE BY THE NO RELEASER, S-NITROSOGLUTATHIONE - NITROSATION OF HOMOCYSTEINE AND MODULATION OF METR BINDING TO THE GLYA-HMP INTERGENIC REGION

The flavohaemoglobin gene, hmp, of Escherichia coil is upregulated by nitric oxide (NO)in a SoxRS-independent manner. We now show that hmp e xpression is also upregulated by S-nitrosoglutathione (GSNO, widely us ed as an NO releaser) and sodium nitroprusside (SNP, which is a NO+ do nor), Elevated homocysteine (Hcy) levels, achieved either by adding He y extracellularly or using metE mutants, decreased hmp expression. Con versely, metC mutants (defective in Hey synthesis) had higher levels o f hmp expression. Mutations in metR abolished hmp induction by GSNO an d SNP, and hmp expression became insensitive to Hey. We propose that t he previously documented modulation by Hey of MetR binding to the glyA -hmp intergenic regulatory region regulates hmp transcription. Althoug h two MetR binding sites are present in this region, only the higher a ffinity site proximal to hmp is required for hmp induction by GSNO and SNP, GSNO and SNP react with Hey in vitro under physiologically relev ant conditions of pH and temperature generating S-nitrosohomocysteine, although in the latter case this would be co-ordinated to the Fe in S NP as a stable species, The free S-nitrosocysteine generated in the re action with GSNO breaks down to release NO more readily than via homol ysis of GSNO, As GSNO and SNP upregulate hmp similarly, the NO release d in the former case on reaction with homocysteine cannot be involved in hmp regulation.