Extended supercomplex contains type-II NADH dehydrogenase, cytochrome bcc complex, and aa3 oxidase in the respiratory chain of Corynebacterium glutamicum.

To clarify the precise subunit composition of the respiratory supercomplex of Corynebacterium glutamicum, several wash conditions were examined. MEGA (9 + 10) wash-buffer (0.5%) was used for this purpose and two-step column chromatography was performed. Almost equal amounts of cytochrome c, b, and a were observed in the purified fraction, estimated by their different absorption spectra. The 833 kDa and 685 kDa bands were observed in the clear native polyacrylamide gel electrophoresis (CN-PAGE) of the purified fraction. Both bands were stained using N,N',N',N-tetramethyl-p-phenylenediamine (TMPD) oxidase dye, and the 833 kDa band was also stained using NADH oxidase dye. The 3D map reconstructed from the 833 kDa band indicated that the bcc complex and aa3 oxidase are heterodimers. Lastly, electron transfer from NADH to the bcc-aa3 supercomplex was observed. The 833 kDa band is the supercomplex, which includes the heterodimer cytochrome bcc complex and cytochrome aa3 oxidase, as well as the monomer NDH-II. Hence, we termed the 833 kDa band the extended supercomplex (ESC).

The cryoEM structure of cytochrome bd from C. glutamicum provides novel insights into structural properties of actinobacterial terminal oxidases.

Cytochromes bd are essential for microaerobic respiration of many prokaryotes including a number of human pathogens. These enzymes catalyze the reduction of molecular oxygen to water using quinols as electron donors. Their importance for prokaryotic survival and the absence of eukaryotic homologs make these enzyme ideal targets for antimicrobial drugs. Here, we determined the cryoEM structure of the menaquinol-oxidizing cytochrome bd-type oxygen reductase of the facultative anaerobic Actinobacterium Corynebacterium glutamicum at a resolution of 2.7 Å. The obtained structure adopts the signature pseudosymmetrical heterodimeric architecture of canonical cytochrome bd oxidases formed by the core subunits CydA and CydB. No accessory subunits were identified for this cytochrome bd homolog. The two b-type hemes and the oxygen binding heme d are organized in a triangular geometry with a protein environment around these redox cofactors similar to that of the closely related cytochrome bd from M. tuberculosis. We identified oxygen and a proton conducting channels emerging from the membrane space and the cytoplasm, respectively. Compared to the prototypical enzyme homolog from the E. coli, the most apparent difference is found in the location and size of the proton channel entry site. In canonical cytochrome bd oxidases quinol oxidation occurs at the highly flexible periplasmic Q-loop located in the loop region between TMHs six and seven. An alternative quinol-binding site near heme b 595 was previously identified for cytochrome bd from M. tuberculosis. We discuss the relevance of the two quinol oxidation sites in actinobacterial bd-type oxidases and highlight important differences that may explain functional and electrochemical differences between C. glutamicum and M. tuberculosis. This study expands our current understanding of the structural diversity of actinobacterial and proteobacterial cytochrome bd oxygen reductases and provides deeper insights into the unique structural and functional properties of various cytochrome bd variants from different phylae.

Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms.

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.

Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases.

The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper-containing oxidases, even though the structures are completely different.

Monitoring the enzyme expression in a respiratory chain of Corynebacterium glutamicum in a copper ion-supplemented culture medium.

Corynebacterium glutamicum has a branched respiratory chain: one of the branches is cytochrome bcc complex and cytochrome aa3-type cytochrome c oxidase, and the other is cytochrome bd-type menaquinol oxidase. The factors that influence the expression patterns of these respiratory enzymes remain unclear. To investigate the expressional control mechanism of the enzymes, we have previously constructed a promoter assay system utilizing enhanced green fluorescence protein. Here, we monitored respiratory enzymes' expression by using this system during growth in various culture media, with and without Cu(2+) ion supplementation. The promoter activities of cytochrome aa3 oxidase in the early stationary phase in the media supplemented with Cu(2+) ion at 40 or 400 µM were significantly increased 1.49-fold or 1.99-fold, respectively, as compared to the control. Moreover, the H(+)/O ratio, or the proton-pumping activity of the cells, increased about 1.6 times by the Cu(2+) supplementation. These facts indicate that copper ions can switch the branches.

Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3.

Several bacteria possess membrane-bound dehydrogenases other than cytosolic dehydrogenases in their respiratory chains. In many cases, the membrane-bound malate:quinone oxidoreductases (MQOs) are essential for growth. However, these MQOs are absent in mammalian mitochondria, and therefore may be a potential drug target for pathogenic bacteria. To characterize the kinetic properties of MQOs, we purified MQO from Bacillus sp. PS3, which is a gram-positive and thermophilic bacterium, and cloned the gene encoding MQO based on the obtained partial N-terminus sequence. Purified MQOs showed a molecular mass of ~90 kDa, which was estimated using gel filtration, and it consists of two subunits with a molecular mass of ~50 kDa. Phylogenetic analysis showed a high similarity to the MQO of the Geobacillus group rather than the Bacillus group. Additionally, the purified enzyme was thermostable and it retained menaquinol reduction activity at high temperatures. Although it is difficult to conduct experiments using menaquinol because of its instability, we were able to measure the oxidase activity of cytochrome bd-type quinol oxidase by using menaquinol-1 by coupling this molecule with the menaquinol reduction reaction using purified MQOs.

Monitoring enzyme expression of a branched respiratory chain of corynebacterium glutamicum using an EGFP reporter gene.

To investigate the expressional control of branched respiratory chain complexes of the amino-acid producing bacterium Corynebacterium glutamicum according to growth conditions, the expression indexes of the ndh, sdh, qcrCAB, ctaCF, ctaD, ctaE, and cydAB genes were estimated under aerobic and microaerobic, and carbon-rich and -poor conditions. The promoter region of each target gene was cloned upstream of the EGFP gene on expression vector pVK6, and the nine reporter constructs were transformed into C. glutamicum ssp. lactofermentum. The cytochrome content of cellular membranes obtained from each growth phase closely corresponded to the expression indexes based on EGFP fluorescence and cell density, indicating that this rapid and convenient method is suitable for analyzing the expression levels of respiratory chain complexes. Using this method, we demonstrated that a reciprocal change in the expression levels of cytochrome bd-type and aa (3)-type oxidases occurs when C. glutamicum cells are held in stationary phase for extended periods.