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

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 0.24 μM) 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. General significance: 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.

Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition

“…The rationale for the latter observation could be that, based on the available literature, beyond its role in cell bioenergetics, cytochrome bd seems to accomplish several important physiological functions that enable bacterial survival in different ecological niches and, importantly, resistance to the hostile conditions created by the immune system in response to microbial infections (reviewed in [14,41,42]). In agreement with this hypothesis, there is growing evidence in the literature pointing to a protective role of cytochrome bd against a number of stressors, including H 2 O 2 [23,[42][43][44][45][46][47][48][49][50][51][52], NO• [41,[53][54][55][56][57][58], CO [59], nitrite [60,61] and excess O 2 itself [62]. Intriguingly, all these species are implicated in the host immune response against microbial infections.…”

Gasotransmitters, poisons, and antimicrobials: it's a gas, gas, gas!

“…Subsequent time-resolved analysis revealed that the transcription of appBC , which encodes cytochrome bd -II is not affected in response to CORM-3 [81]. In support, a strain expressing cytochrome bd -I as the sole oxidase is less susceptible to both respiratory and growth inhibition by this compound [82], consistent with the established role of this oxidase in resisting a variety of environmental stresses including NO [83]. In the case of NO, it has been proposed that cytochrome bd resists inhibition because of an exceptionally high off rate ( k off 0.163 s −1 for cytochrome bd , compared to 0.03 s −1 for cytochrome bo' [83]), although it is difficult to draw such conclusions for CO due to discrepancies in the literature as to the off rate of this gas from cytochrome bd -I (see references in [82]).…”

Interaction of the carbon monoxide-releasing molecule Ru(CO)3Cl(glycinate) (CORM-3) with Salmonella enterica serovar Typhimurium: in situ measurements of carbon monoxide binding by integrating cavity dual-beam spectrophotometry

“…bactericidal activity of CO-RMs is reduced by the presence of a number of molecules, including N-acetylcysteine, cysteine and reduced glutathione (Desmard et al, 2009;Jesse et al, 2013;McLean et al, 2013), and so is likely to be diminished in a complex medium such as LB. The reasons are unclear.…”