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

Elena Forte,Alessandro Giuffrè,Vitaliy B Borisov,Li-Shar Huang,Edward A Berry

doi:10.3390/ijms21228521

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 smegmatis aa3-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.

Highlights

Nitric oxide (NO) is a gaseous free radical that, while exerting physiological functions at low concentrations, can have deleterious effects on the cell at high levels, being able itself or in combination with reactive oxygen species to damage proteins, lipids and nucleic acids
We investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatis aa3-type terminal oxidase
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−1 min−1 at 30 μM NO

Summary

Introduction

Nitric oxide (NO) is a gaseous free radical that, while exerting physiological functions at low concentrations, can have deleterious effects on the cell at high levels, being able itself or in combination with reactive oxygen species to damage proteins, lipids and nucleic acids. Mycobacteria have no soluble cytochrome c, but the bcc complex displays a dihaem c-type cytochrome playing the role of the cytochromes c and c1 in canonical electron transfer chains. The bcc complex encoded by the qcrCAB operon transfers electrons from menaquinol to the aa3-type cytochrome c oxidase. It comprises cytochrome b (QcrB) containing two b haem groups, a Rieske-type high potential Fe2S2 iron-sulfur protein (QcrA), and a dihaem c-type cytochrome (QcrC). Like other haem-copper terminal oxidases [19,20,21,22,23,24,25,26,27], the mycobacterial bcc-aa supercomplex is proposed to act as a proton pump to generate the proton-motive force [16]. We found that the enzyme resists inhibition by the ligand, and rapidly metabolizes it

Results

Supercomplex Purification and Concentration Determination

Catalytic Assays

Data Analysis