MOESM3 of Pseudomonas stutzeri as an alternative host for membrane proteins

Additional file 3: Table S3. Production level of all tested constructs at different conditions

Resonance Raman Characterization of the Ammonia-Generated Oxo Intermediate of Cytochrome <i>c</i> Oxidase from <i>Paracoccus denitrificans</i>

A novel oxo state of cytochrome <i>c</i> oxidase from <i>Paracoccus denitrificans</i> generated by successive addition of excess H₂O₂ and ammonia was investigated using resonance Raman (RR) spectroscopy. Addition of ammonia to the H₂O₂-generated artificial F state resulted in an upshift of the oxoferryl stretching vibration from 790 to 796 cm^–1, indicating that ammonia influences ligation of the heme-bound oxygen in the binuclear center. Concomitantly performed RR measurements in the high-frequency region between 1300 and 1700 cm^–1 showed a high-spin to low-spin transition of heme <i>a</i>₃ upon generation of the F state that was not altered by addition of ammonia. Removal of H₂O₂ by addition of catalase resulted in the disappearance of the oxoferryl stretching vibration and major back transformation of heme <i>a</i>₃ into the high-spin state. The ratio of high-spin to low-spin states was identical for intermediates created with and without ammonia, leading to the conclusion that ammonia does not interact directly with heme <i>a</i>₃. Only for the ammonia-created state was a band at 612 nm observed in the UV–visible difference spectrum that was shifted to 608 nm after addition of catalase. Our results support the hypothesis by von der Hocht et al. [von der Hocht, I., et al. (2011) <i>Proc. Natl. Acad. Sci. U.S.A. 108</i>, 3964–3969] that addition of ammonia creates a novel oxo intermediate state called P_N where ammonia binds to Cu_B once the oxo intermediate F state has been formed

DataSheet1_The cryoEM structure of cytochrome bd from C. glutamicum provides novel insights into structural properties of actinobacterial terminal oxidases.docx

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 b595 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.</p