Fast Cytochrome bo from Escherichia coli Binds Two Molecules of Nitric Oxide at Cu_B

Abstract

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.

Response ofBacillus subtilisto Nitric Oxide and the Nitrosating Agent Sodium Nitroprusside

“…Because NO inhibits aerobic respiration under similar conditions (Fig. 2), we suggest that depressed ATP levels may trigger the B response via the energy stress branch (10,29). Unexpectedly, SNP activated B induction through the environmental rather than the energy stress pathway and induction was noted both aerobically and, to a lesser extent, anaerobically (Fig.…”

Nitric Oxide in Chemostat-Cultured Escherichia coli Is Sensed by Fnr and Other Global Regulators: Unaltered Methionine Biosynthesis Indicates Lack of S Nitrosation

“…Recently, however, Mason et al (43) have shown that NO inhibition of respiration involves both competitive (at the heme) and noncompetitive (at the copper) binding to cytochrome c oxidase. Significantly, cytochrome bd contains no copper (36), whereas the cytochrome boЈ quinol oxidase possesses a heme-Cu B active site like that of cytochrome c oxidase, and the copper atom is capable of binding NO (9). Cytochrome bd is widely considered to confer tolerance to a number of apparently unrelated cellular stresses, including metal ions, cyanide, and reductants (62).…”

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

“…The cis mechanism has been strongly favored by Thomson and coworkers because it does not involve the formation of a heme b {FeNO} 7 species, which they view as a potential dead-end product. [62][63][64][65] Indeed, in many hemoproteins, ferrous-nitrosyl complexes are unreactive species. [66][67][68][69][70] Redox titrations of cNOR by Thomson and coworkers have indicated that the redox potential of heme b 3 is 200 mV more negative than that of the non-heme iron Fe B , suggesting that a three-electron reduced state, where heme b 3 remains oxidized, may be relevant to catalysis.…”