Biochemical and Biophysical Characterization of the Two Isoforms of cbb3-Type Cytochrome c Oxidase from Pseudomonas stutzeri

Abstract

The cbb 3 -type cytochrome c oxidases (cbb 3 -CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb 3 -CcOs are present only in bacteria and play a primary role in microaerobic respiration, being essential for nitrogen-fixing endosymbionts and for some human pathogens. As frequently observed in Pseudomonads, Pseudomonas stutzeri contains two independent ccoNO(Q)P operons encoding the two cbb 3 isoforms, Cbb 3 -1 and Cbb 3 -2. While the crystal structure of Cbb 3 -1 from P. stutzeri was determined recently and cbb 3 -CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb 3 -CcOs. In this work, both isoforms were homologously expressed in P. stutzeri strains from which the genomic version of the respective operon was deleted. We purified both cbb 3 isoforms separately by affinity chromatography and increased the yield of Cbb 3 -2 to a similar level as Cbb 3 -1 by replacing its native promoter. Mass spectrometry, UV-visible (UV-Vis) spectroscopy, differential scanning calorimetry, as well as oxygen reductase and catalase activity measurements were employed to characterize both cbb 3 isoforms. Differences were found concerning the thermal stability and the presence of subunit CcoQ. However, no significant differences between the two isoforms were observed otherwise. Interestingly, a surprisingly high turnover of at least 2,000 electrons s ؊1 and a high Michaelis-Menten constant (K m ϳ 3.6 mM) using ascorbate-N,N,N=,N=-tetramethyl-pphenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb 3 -CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb 3 isoforms specifically.

Regulation of nitrite resistance of the cytochrome cbb3 oxidase by cytochrome c ScyA in Shewanella oneidensis

“…; Xie et al. ). The IC 50 values of the cytochrome bd and cbb 3 ‐HCO for nitrite were obtained from plots of rates against nitrite concentrations.…”

Enzymatic Characterization and In Vivo Function of Five Terminal Oxidases in Pseudomonas aeruginosa

“…Recently, Xie et al (45) purified and characterized the two cbb 3 isoforms from P. stutzeri and found that they …”

Expression of multiplecbb3cytochromecoxidase isoforms by combinations of multiple isosubunits inPseudomonas aeruginosa

“…The isoforms corresponding to cbb 3 -1 and cbb 3 -2 of P. aeruginosa are oppositely designated as cbb 3 -2 and cbb 3 -1, respectively, in other Pseudomonas species, such as Pseudomonas putida and P. stutzeri (21,22). Xie et al reported that the two isoforms from P. stutzeri differed in thermal stability but had no significant difference regarding the UV-visible spectrum or enzymatic activities (22). The affinities of the N1-and N2-type isoforms for oxygen were not significantly different in P. aeruginosa or the difference was smaller than the experimental errors (Table 1).…”