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The haem b558 component of the cytochrome bd quinol oxidase complex from Escherichia coli has histidine-methionine axial ligation.

ABSTRACT: The cytochrome bd ubiquinol oxidase from Escherichia coli is induced when the bacteria are cultured under microaerophilic or low-aeration conditions. This membrane-bound respiratory oxidase catalyses the two-electron oxidation of ubiquinol and the four-electron reduction of dioxygen to water. The oxidase contains three haem prosthetic groups: haem b558, haem b595 and haem d. Haem d is the oxygen binding site, and it is likely that haem d and b595 form a bimetallic site in the enzyme. Haem b558 has been previously characterized spectroscopically as being low spin and has been shown to be located within subunit I (CydA) of this two-subunit enzyme. It is likely that haem b558 is associated with the quinol oxidation site, which has also been shown to be within subunit I. In a previous effort to locate the specific amino acids axially ligated to haem b558, all six histidines within subunit I were altered by site-directed mutagenesis. Only one, histidine-186, was identified as a likely ligand to haem b558. Hence it was suggested that haem b558 could not have bis(histidine) ligation. In the current work, a combination of low-temperature near-infrared magnetic circular dichroism (NIR-MCD) and EPR spectroscopies have been employed to identify the nature of the haem b558 axial ligands. The NIR-MCD spectrum at cryogenic temperatures is dominated by the low-spin haem b558 component of the complex, and the low-energy band near 1800 nm is strong evidence for histidine-methionine ligation. It is concluded that haem b558 is ligated to histidine-186 plus one of the methionines located within subunit I of the oxidase.

SUBMITTER: Spinner F

PROVIDER: S-EPMC1136974 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Project description:Cytochrome bd quinol oxidases, which have a greater affinity for oxygen than heme-copper cytochrome oxidases (HCOs), promote bacterial respiration and fitness in low-oxygen environments, such as host tissues. Here, we show that, in addition to the CydA and CydB subunits, the small protein CydX is required for the assembly and function of the cytochrome bd complex in the enteric pathogen Salmonella enterica serovar Typhimurium. Mutant S Typhimurium lacking CydX showed a loss of proper heme arrangement and impaired oxidase activity comparable to that of a ΔcydABX mutant lacking all cytochrome bd subunits. Moreover, both the ΔcydX mutant and the ΔcydABX mutant showed increased sensitivity to β-mercaptoethanol and nitric oxide (NO). Cytochrome bd-mediated protection from β-mercaptoethanol was not a result of resistance to reducing damage but, rather, was due to cytochrome bd oxidase managing Salmonella respiration, while β-mercaptoethanol interacted with the copper ions necessary for the HCO activity of the cytochrome bo-type quinol oxidase. Interactions between NO and hemes in cytochrome bd and cytochrome bd-dependent respiration during nitrosative stress indicated a direct role for cytochrome bd in mediating Salmonella resistance to NO. Additionally, CydX was required for S Typhimurium proliferation inside macrophages. Mutants deficient in cytochrome bd, however, showed a significant increase in resistance to antibiotics, including aminoglycosides, d-cycloserine, and ampicillin. The essential role of CydX in cytochrome bd assembly and function suggests that targeting this small protein could be a useful antimicrobial strategy, but potential drug tolerance responses should also be considered.IMPORTANCE Cytochrome bd quinol oxidases, which are found only in bacteria, govern the fitness of many facultative anaerobic pathogens by promoting respiration in low-oxygen environments and by conferring resistance to antimicrobial radicals. Thus, cytochrome bd complex assembly and activity are considered potential therapeutic targets. Here we report that the small protein CydX is required for the assembly and function of the cytochrome bd complex in S Typhimurium under stress conditions, including exposure to β-mercaptoethanol, nitric oxide, or the phagocytic intracellular environment, demonstrating its crucial function for Salmonella fitness. However, cytochrome bd inactivation also leads to increased resistance to some antibiotics, so considerable caution should be taken when developing therapeutic strategies targeting the CydX-dependent cytochrome bd.

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The haem b558 component of the cytochrome bd quinol oxidase complex from Escherichia coli has histidine-methionine axial ligation.

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