The roles of the hybrid cluster protein, Hcp and its reductase, Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress

Abstract

The hybrid cluster protein, Hcp, contains a 4Fe-2S-2O iron-sulfur-oxygen cluster that is currently considered to be unique in biology. It protects various bacteria from nitrosative stress, but the mechanism is unknown. We demonstrate that the Escherichia coli Hcp is a high affinity nitric oxide (NO) reductase that is the major enzyme for reducing NO stoichiometrically to N2 O under physiologically relevant conditions. Deletion of hcp results in extreme sensitivity to NO during anaerobic growth and inactivation of the iron-sulfur proteins, aconitase and fumarase, by accumulated cytoplasmic NO. Site directed mutagenesis revealed an essential role in NO reduction for the conserved glutamate 492 that coordinates the hybrid cluster. The second gene of the hcp-hcr operon encodes an NADH-dependent reductase, Hcr. Tight interaction between Hcp and Hcr was demonstrated. Although Hcp and Hcr purified individually were inactive or when recombined, a co-purified preparation reduced NO in vitro with a Km for NO of 500 nM. In an hcr mutant, Hcp is reversibly inactivated by NO concentrations above 200 nM, indicating that Hcr protects Hcp from nitrosylation by its substrate, NO.

13. Reactivity of iron-sulfur clusters with nitric oxide

“…In E. coli under anaerobic conditions, in the presence of nitrate, NO appears to be maintained well below 1 µM [148]. The activation of NorR occurs at an NO concentration approximately 10-fold higher than the NO induced de-repression of NsrR controlled genes, such as hcp [148]. Moreover, FNR-dependent expression drops significantly at extracellular concentrations of ≥5 µM, suggesting an upper limit for the detoxifying capacity of the first line detoxification systems [65].…”

“…We note that Hcp, by virtue of its iron-sulfur clusters, is susceptible to nitrosylation at elevated NO levels, although the physiological concentration of NO has been hotly contested. In E. coli under anaerobic conditions, in the presence of nitrate, NO appears to be maintained well below 1 µM [148]. The activation of NorR occurs at an NO concentration approximately 10-fold higher than the NO induced de-repression of NsrR controlled genes, such as hcp [148].…”

“…The role Hcp plays in the nitrosoative stress response during anaerobic growth has been something of an enigma since its discovery. However, recent work by Wang et al [148] has shown that Hcp functions along with an NADH-dependent reductase, Hcr, as a high affinity NO reductase that stoichometrically reduces NO to N2O. These discoveries led to the proposal that Hcp-Hcr is the major NO reductase at very low concentrations of NO (which are commonly found in the bacterial cytoplasm during the anaerobic reduction of nitrate and nitrite).…”

“…In contrast, Hcp expression is strongly induced under anaerobic conditions by FNR and is repressed by NsrR, consistent with its role as an NO reductase. Hcp is discussed in more detail later on because it is an Fe-S cluster protein that reacts with NO in a thus far unique way [148].…”

“…(b) Fine detail of the catalytic hybrid cluster (from PDB: 1E1D) showing ligand arrangement, oxygen atoms and cysteine persulfide (on C406). The hybrid cluster catalyzes the reduction of NO to nitrous oxide N2O [148].…”