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Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa₃ Supercomplex.

ABSTRACT: Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa₃ supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa₃-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O₂ and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ?303 mol NO (mol enzyme)^-1 min^-1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O₂ consumption, with 2.65 ± 0.19 molecules of NO being consumed per O₂ molecule by the mycobacterial bcc-aa₃. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)^-1 min^-1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa₃ supercomplexes against NO stress.

SUBMITTER: Forte E

PROVIDER: S-EPMC7697965 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex.

Forte Elena E Giuffrè Alessandro A Huang Li-Shar LS Berry Edward A EA Borisov Vitaliy B VB

International journal of molecular sciences 20201112 22

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able t ...[more]

PMID: 33198276

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Project description:Spores have strongly reduced metabolic activity and are produced during the complex developmental cycle of the actinobacterium Streptomyces coelicolor Resting spores can remain viable for decades, yet little is known about how they conserve energy. It is known, however, that they can reduce either oxygen or nitrate using endogenous electron sources. S. coelicolor uses either a cytochrome bd oxidase or a cytochrome bcc-aa 3 oxidase supercomplex to reduce oxygen, while nitrate is reduced by Nar-type nitrate reductases, which typically oxidize quinol directly. Here, we show that in resting spores the Nar1 nitrate reductase requires a functional bcc-aa 3 supercomplex to reduce nitrate. Mutants lacking the complete qcr-cta genetic locus encoding the bcc-aa 3 supercomplex showed no Nar1-dependent nitrate reduction. Recovery of Nar1 activity was achieved by genetic complementation but only when the complete qcr-cta locus was reintroduced to the mutant strain. We could exclude that the dependence on the supercomplex for nitrate reduction was via regulation of nitrate transport. Moreover, the catalytic subunit, NarG1, of Nar1 was synthesized in the qcr-cta mutant, ruling out transcriptional control. Constitutive synthesis of Nar1 in mycelium revealed that the enzyme was poorly active in this compartment, suggesting that the Nar1 enzyme cannot act as a typical quinol oxidase. Notably, nitrate reduction by the Nar2 enzyme, which is active in growing mycelium, was not wholly dependent on the bcc-aa 3 supercomplex for activity. Together, our data suggest that Nar1 functions together with the proton-translocating bcc-aa 3 supercomplex to increase the efficiency of energy conservation in resting spores.IMPORTANCE Streptomyces coelicolor forms spores that respire with either oxygen or nitrate, using only endogenous electron donors. This helps maintain a membrane potential and, thus, viability. Respiratory nitrate reductase (Nar) usually receives electrons directly from reduced quinone species; however, we show that nitrate respiration in spores requires a respiratory supercomplex comprising cytochrome bcc oxidoreductase and aa 3 oxidase. Our findings suggest that the Nar1 enzyme in the S. coelicolor spore functions together with the proton-translocating bcc-aa 3 supercomplex to help maintain the membrane potential more efficiently. Dissecting the mechanisms underlying this survival strategy is important for our general understanding of bacterial persistence during infection processes and of how bacteria might deal with nutrient limitation in the natural environment.

Dataset Information

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa₃ Supercomplex.

Publications

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome <i>bcc</i>-<i>aa</i><sub>3</sub> Supercomplex.

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