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

The cbb3-type cytochrome c oxidases (cbb3-CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb3-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 cbb3 isoforms, Cbb3-1 and Cbb3-2. While the crystal structure of Cbb3-1 from P. stutzeri was determined recently and cbb3-CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb3-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 cbb3 isoforms separately by affinity chromatography and increased the yield of Cbb3-2 to a similar level as Cbb3-1 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 cbb3 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 (Km ~ 3.6 mM) using ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb3-CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb3 isoforms specifically.