The nitric oxide interactions with erythrocytes and Escherichia coli

Nitric oxide (NO) is a physiologically important signaling molecule and it is endogenously produced. In human, it is unclear how the homeostasis of NO in the vasculature is maintained in the presence of red blood cells (RBCs), which contains concentrated amount of NO-consuming protein, hemoglobin (Hb). Nitrosylhemoglobin (HbFeIINO) has been detected in vivo and its role in NO transport and preservation has been discussed. We performed in vitro experiments to determine the effect of oxygenated red blood cells (RBCs) on the dissociation of cell-free HbFe IINO. Results show that RBC did not change the apparent half-life of extracellular HbFeIINO, but caused a shift in the HbFe IINO dissociation products from methemoglobin (metHbFeIII) to oxyhemoglobin (HbFeIIO2). Although these results appear to suggest some unusual interactions between HbFeIINO and RBC, the data are explainable by HbFeIINO dissociation and hemoglobin oxidation with known rate constants. A kinetic model consisting of these reactions shows that (i) deoxyhemoglobin is an intermediate in the reaction of HbFeIINO oxidation to metHbFeIII, (ii) the rate-limiting step of HbFeIIINO decay is the dissociation of NO from HbFeIINO, (iii) the magnitude of NO diffusion rate constant into RBC is estimated to be ∼104 M-1s -1, consistent with the previous results determined from a competition assay, and (iv) no additional chemical reactions are required to explain these data.;NO is also used by mammalian immune systems to combat microbial invasions. To uncover the comprehensive NO-responsive genes, we screened the library of single-gene knockouts of Escherichia coli for mutants that exhibit resistance to NO. We have discovered that the fre gene deletion confers NO-resistance to E. coli. The NO-sensitivity could also be restored when the mutated fre was repaired or when the fre deletion mutant was transformed with fre cloned. Data strongly indicated the intermediacy of peroxynitrite (ONOO-) and its toxicity, because the addition of sodium bicarbonate rescued the oxygen-dependent NO-mediated growth inhibition. Results suggested that Fre produced superoxide radical which reacts with NO and enhanced the NO-toxicity by forming ONOO-, so the fre deletion confers NO-resistance.