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Biochim Biophys Acta. 2013 Sep;1834(9):1693-703. doi: 10.1016/j.bbapap.2013.04.019. Epub 2013 Apr 26.

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.

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Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, S10 2TN, UK.



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.


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.


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.


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.


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.


(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

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