title: Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo'' to inhibition by the carbon monoxide-releasing molecule, CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration.
abstract: BACKGROUND
  CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO - a critical gasotransmitter - in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident.
  
  
  METHODS
  Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli - cytochrome bd-I, cytochrome bd-II and cytochrome bo', to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood.
  
  
  RESULTS
  We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo'. Cytochromes bo' and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells.
  
  
  CONCLUSIONS
  A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli.
  
  
  CONCLUSIONS
  Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
authors.affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, S10 2TN, UK. mpb09hej@sheffield.ac.uk
authors.forename: Helen E
authors.initials: HE
authors.lastname: Jesse
authors.forename: Tacita L
authors.initials: TL
authors.lastname: Nye
authors.forename: Samantha
authors.initials: S
authors.lastname: McLean
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.forename: Brian E
authors.initials: BE
authors.lastname: Mann
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D002248:Carbon Monoxide / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D045222:Electron Transport Chain Complex Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006931:Hyperbaric Oxygenation; D007874:Leghemoglobin / Q000378:metabolism; D009942:Organometallic Compounds / Q000494:pharmacology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects*; D012428:Ruthenium / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D007874:Leghemoglobin; D009942:Organometallic Compounds; C095131:oxyleghemoglobin; C476530:tricarbonylchloro(glycinato)ruthenium(II); D006418:Heme; D002248:Carbon Monoxide; D012428:Ruthenium; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D000111:Acetylcysteine
keywords: (2′7′-diacetate); ([Ru(CO)(3)Cl(2)](2)); CO-RM; CORM-2; CORM-3; Cytochrome; DCFH-DA; EDTA; EGTA; Escherichia coli; HO-1; ICP-MS; K(L)a; K(d); K(m); LB; Luria Bertani broth; Michaelis constant, the concentration of substrate that gives half-maximal velocity; N-acetylcysteine; NAC; PBS; ROS; Respiratory oxidase; Ru(CO)(3)Cl(glycinate); SOD; V(max); carbon monoxide-releasing molecule; dissociation constant; ethylene diamine tetraacetic acid; ethylene glycol tetraacetic acid; gas transfer (gas to liquid) coefficient; haem oxygenase-1; iCORM-3; inactive CORM-3; inductively coupled plasma mass spectrometry; maximal rate; miCORM-3; myoglobin-inactivated CORM-3; phosphate-buffered saline; reactive oxygen species; superoxide dismutase