Molecular and Spectroscopic Analysis of the Cytochromecbb3 Oxidase from Pseudomonas stutzeri

Abstract

Cytochrome cbb 3 oxidase, a member of the heme-copper oxidase superfamily, is characterized by its high affinity for oxygen while retaining the ability to pump protons. These attributes are central to its proposed role in the microaerobic metabolism of proteobacteria. We have completed the first detailed spectroscopic characterization of a cytochrome cbb 3 oxidase, the enzyme purified from Pseudomonas stutzeri. A combination of UVvisible and magnetic CD spectroscopies clearly identified four low-spin hemes and the high-spin heme of the active site. This heme complement is in good agreement with our analysis of the primary sequence of the ccoNOPQ operon and biochemical analysis of the complex. Near-IR magnetic CD spectroscopy revealed the unexpected presence of a low-spin bishistidine-coordinated c-type heme in the complex. This was shown to be one of two c-type hemes in the CcoP subunit by separately expressing the subunit in Escherichia coli. Separate expression of CcoP also allowed us to unambiguously assign each of the signals associated with low-spin ferric hemes present in the X-band EPR spectrum of the oxidized enzyme. This work both underpins future mechanistic studies on this distinctive class of bacterial oxidases and raises questions concerning the role of CcoP in electron delivery to the catalytic subunit.

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

“…As shown in Fig. 4A, the cbb 3 -CcO was found to be most active at pH 7.5, which is consistent with a previous report (26). By varying the concentration of NaCl in the range of 0 to 500 mM, the inter- action between TMPD and cbb 3 -CcO appears to be independent of the ionic strength (Fig.…”

“…A DNA sequence comparison showed that the first cbb 3 operon (cco-NOP-1) has, on average, a 79% identity with ccoNOQP-2, which might explain why only one operon was found previously (26). The amino acid sequence identities of the two cbb 3 isoforms are also very high, 87% for subunit CcoN, 97% for subunit CcoO, and 63% for subunit CcoP.…”

“…Earlier, P. stutzeri strain ZoBell was reported to possess only one ccoNOQP operon coding for cbb 3 -CcO (26). More recently, we showed that this strain actually possesses two independent cbb 3 operons, encoding isoforms of cbb 3 -CcO as well as Cbb 3 -1 and Cbb 3 -2 (19) (GenBank accession number HM130676).…”

“…Cbb 3 -CcOs were oxidized with 10-fold molar excess of potassium hexacyanoferrate(III) and fully reduced by adding a small amount of sodium dithionite. The concentrations of oxidized cbb 3 -CcOs were calculated using a molar extinction coefficient of 5.85 ϫ 10 5 M Ϫ1 cm Ϫ1 at 411 nm (26). Differential scanning calorimetry.…”

Use of surface‐enhanced laser desorption/ionization ‐time of flight to explore bacterial proteomes

“…These papers mainly describe the use of the ProteinChip Arrays with purified proteins [10][11][12][13][14], or extracted surface proteins [15][16][17][18][19]. A few reports are available in the literature in which this technology is used with protein extracts [20][21][22].…”