Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix

<p>Abstract</p> <p>Background</p> <p>The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical <it>bc</it>₁complex has not yet been isolated from Archaea. In particular, <it>c</it>-type cytochromes have been reported only for a limited number of species.</p> <p>Results</p> <p>Here, we isolated a <it>c</it>-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, <it>Aeropyrum pernix</it>, grown aerobically. The redox spectrum of the isolated <it>c</it>-type cytochrome showed a characteristic α-band peak at 553 nm corresponding to heme C. The pyridine hemochrome spectrum also revealed the presence of heme B. In non-denaturing polyacrylamide gel electrophoresis, the cytochrome migrated as a single band with an apparent molecular mass of 80 kDa, and successive SDS-PAGE separated the 80-kDa band into 3 polypeptides with apparent molecular masses of 40, 30, and 25 kDa. The results of mass spectrometry indicated that the 25-kDa band corresponded to the hypothetical cytochrome <it>c </it>subunit encoded by the ORF <it>APE_1719.1</it>. In addition, the <it>c</it>-type cytochrome-containing polypeptide complex exhibited menaquinone: yeast cytochrome <it>c </it>oxidoreductase activities.</p> <p>Conclusion</p> <p>In conclusion, we showed that <it>A. pernix</it>, a hyperthemophilic archaeon, has a "full" <it>bc </it>complex that includes a <it>c</it>-type cytochrome, and to the best of our knowledge, <it>A. pernix </it>is the first archaea from which such a <it>bc </it>complex has been identified. However, an electron donor candidates for cytochrome <it>c </it>oxidase, such as a blue copper protein, have not yet been identified in the whole genome data of this archaeon. We are currently trying to identify an authentic substrate between a <it>bc </it>complex and terminal oxidase.</p

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

AbstractWe constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo3-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus.B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo3-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H+ pumping activity, although the efficiency was lower than those of cytochrome aa3-type oxidases belonging to the SoxM-type

Correlation Between Proton Translocation and Growth: Genetic Analysis of the Respiratory Chain of Corynebacterium glutamicum

Corynebacterium glutamicum contains at least two terminal oxidases in the respiratory chain; cytochrome aa3-type cytochrome c oxidase and bd-type menaquinol oxidase. Thus, the chain has two branches of electron flow. The bcc-aa3 branch translocates three protons per electron transferred, while the bd branch translocates only one. In this study, we constructed two mutant strains, lacking of either subunit I of the cytochrome c oxidase (ΔctaD) or subunits I and II of the quinol oxidase (ΔcydAB), and also plasmids to complement the deficient genes to investigate their effects on energy conservation and cell growth. We measured H+/O ratios of C. glutamicum wild-type and mutant cells grown aerobically. The H+/O ratio of the wild-type cells grown in the semi-synthetic medium was 3.94 ± 0.30, while the value was 2.76 ± 0.25 for the ΔctaD mutant. In contrast, the value was 5.23 ± 0.36 for the ΔcydAB mutant. The cells grown in the LB medium showed higher value compared to that of cells grown in the semi-synthetic medium. The ΔctaD mutant grew less than the wild-type in LB medium, while they grew about equally in semi-synthetic medium. Correlation between bioenergetics and growth of C. glutamicum was significantly affected by the growth nutrients

Mutation Analysis of the Interaction of B-type Cytochrome c Oxidase with its Natural Substrate Cytochrome c-551

Heme-copper oxidases in the respiratory chain are classified into three subfamilies: A-, B- and C-types. Cytochrome bo3-type cytochrome c oxidase from thermophilic Bacillus is a B-type oxidase that is thought to interact with cytochrome c through hydrophobic interactions. This is in contrast to A-type oxidases, which bind cytochrome c molecules primarily through electrostatic forces between acidic residues in the oxidase subunit II and basic residues within cytochromes. In order to investigate the substrate-binding site in cytochrome bo3, eight acidic residues in subunit II were mutated to corresponding neutral residues and enzymatic activity was measured using cytochrome c-551 from closely related Bacillus PS3. The mutation of E116, located at the interface to subunit I, decreased the kcat value most prominently without affecting the Km value, indicating that the residue is important for electron transfer. The mutation of D99, located close to the CuA site, largely affected both values, suggesting that it is important for both electron transfer and substrate binding. The mutation of D49 and E84 did not affect enzyme kinetic parameters, but the mutation of E64, E66 and E68 lowered the affinity of cytochrome bo3 for cytochrome c-551 without affecting the kcat value. These three residues are located at the front of the hydrophilic globular domain and distant from the CuA site, suggesting that these amino acids compose an acidic patch for a second substrate-binding site. This is the first report on site-directed mutagenesis experiments of a B-type heme-copper oxidase

The cytochrome bcc-aa3-type respiratory chain of Rhodococcus rhodochrous

Rhodococcus rhodochrous is an active soil bacterium belonging to the Nocardia group of high GC Gram-positive bacteria. It is rich in various enzymes and thus important in the industrial production of chemicals and bioremediation. In this work, the respiratory chain of this aerobic organism was investigated and characterized. Grown under highly aerobic conditions, the membrane fraction of R. rhodochrous cells only contained a-, b- and c-type cytochromes, suggesting that it is the cytochrome bcc-aa3-type pathway that mainly operates under these conditions. In contrast, the d-type cytochrome was also present under microaerobic conditions, indicating that the alternative pathway of the bd-type oxidase works in these circumstances. In addition, the results of H+/O ratio measurements indicate that these two pathways have different energy efficiencies