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.
Synthesis of the Escherichia coli YtfE protein, also known as RIC, for the repair of damaged iron centres, is highly induced during anaerobic growth under conditions of nitrosative stress. How YtfE repairs nitrosative damage remains unclear. Contrary to previous reports, we show that strains defective in YtfE that lack the high-affinity NO reductase activity of the hybrid cluster protein (Hcp) are less sensitive to nitrosative stress than isogenic ytfE strains, which are extremely sensitive. Evidence that this sensitivity is due to YtfE-dependent release of NO into the cytoplasm includes: relief of growth inhibition by PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), which degrades NO; relief of nitrosative stress by deletion of narG encoding the nitrate reductase that is the major source of NO from nitrite; partial suppression of nitrosative stress due to loss of Hcp function by a further mutation in ytfE; YtfE-dependent loss of aconitase and fumarase activities in the absence of Hcp; and YtfE-dependent relief of NsrR repression of the hcp promoter in response to cytoplasmic NO. We suggest that a major role for YtfE is to reverse nitrosative damage by releasing, directly or indirectly, NO from nitrosylated proteins into the cytoplasm where the high-affinity NO reductase activity of Hcp ensures its reduction to N2O. If so, the concerted action of YtfE and Hcp would not only maintain the cytoplasmic concentration of NO in the low nM range, but also provide a rationalization for the coordinate regulation of Hcp and YtfE synthesis by NsrR.
Previously characterized
nitrite reductases fall into three classes:
siroheme-containing enzymes (NirBD), cytochrome
c
hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK).
We show here that the di-iron protein YtfE represents a physiologically
relevant new class of nitrite reductases. Several functions have been
previously proposed for YtfE, including donating iron for the repair
of iron–sulfur clusters that have been damaged by nitrosative
stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing
NO to nitrous oxide (N
2
O). Here,
in vivo
reporter assays confirmed that
Escherichia coli
YtfE increased cytoplasmic NO production from nitrite. Spectroscopic
and mass spectrometric investigations revealed that the di-iron site
of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound,
when isolated from nitrite-supplemented, but not nitrate-supplemented,
cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated
YtfE and the release of NO. Kinetics of nitrite reduction were dependent
on the nature of the reductant; the lowest
K
m
, measured for the di-ferrous form, was ∼90 μM,
well within the intracellular nitrite concentration range. The vicinal
di-cysteine motif, located in the N-terminal domain of YtfE, was shown
to function in the delivery of electrons to the di-iron center. Notably,
YtfE exhibited very low NO reductase activity and was only able to
act as an iron donor for reconstitution of apo-ferredoxin under conditions
that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity
nitrite reductase that we propose functions to relieve nitrosative
stress by acting in combination with the co-regulated NO-consuming
enzymes Hmp and Hcp.
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