What ATP binding does to the Ca2+ pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2.

Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+ transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates Ca2+ binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+ state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.

Identification of novel inhibitors of human SPCA2.

To identify specific inhibitors of the human secretary pathway Ca(2+)-ATPase 2 (hSPCA2), a recombinant hSPCA2 was expressed in Saccharomyces cerevisiae, and purified by Co(2+)-chelating chromatography. The isolated hSPCA2 catalyzed ATP hydrolysis in the presence of Ca(2+) ions. The Ca(2+) dissociation constant for ATPase activation was 25 nM. hSPCA2 activity was inhibited by thapsigargin, 2,2'-methylenebis(6-tert-butyl-p-cresol), and 4-octylphenol in the low-micromolar concentration range. Unexpectedly, the organic solvent wash from standard laboratory polypropylene microtubes showed strong inhibitory potency toward hSPCA2 activity. The extract was found to comprise mainly primary fatty acid amides (PFAAs) by NMR analysis. Individual PFAAs, especially oleamide and linoleamide, almost completely inhibited hSPCA2 activity with IC50 values of 7.5 µM and 3.8 µM, respectively.

Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans.

Cytochromes bd are terminal oxidases in the respiratory chains of many prokaryotic organisms. They reduce O2 to 2H2O at the expense of electrons extracted from quinol. The oxidases can be divided into two subfamilies, L and S, based on the presence of either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I designated as 'Q-loop'. The L-subfamily members, e.g. the enzyme from Escherichia coli, are relatively well-studied and were shown to generate proton-motive force. The S-subfamily comprises the majority of cytochromes bd including the enzyme from Geobacillus thermodenitrificans but is very poor studied. We compared the properties of cytochromes bd from G. thermodenitrificans and E. coli at room temperature using a combination of absorption, CD and MCD spectroscopy. The G. thermodenitrificans enzyme does contain the high-spin heme b(HS) ("b(595)") despite the fact that its characteristic Q(00)-band ("α"-band) at 595nm is not seen in the absorption spectra; stoichiometry of hemes b(LS), b(HS) and d per the enzyme complex is suggested to be 1:1:1. At 1mM CO, 20-25% of ferrous heme b(HS) in the G. thermodenitrificans oxidase binds the ligand, while in case of the E. coli enzyme such a reaction is minor. In the G. thermodenitrificans oxidase, the excitonic interaction between ferrous hemes b(HS) and d decreased as compared to that in the E. coli bd. The latter may suggest that the two enzymes differ in the distance between heme d and heme b(HS) and/or in the angle between their porphyrin planes.

Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3.

Several bacteria possess membrane-bound dehydrogenases other than cytosolic dehydrogenases in their respiratory chains. In many cases, the membrane-bound malate:quinone oxidoreductases (MQOs) are essential for growth. However, these MQOs are absent in mammalian mitochondria, and therefore may be a potential drug target for pathogenic bacteria. To characterize the kinetic properties of MQOs, we purified MQO from Bacillus sp. PS3, which is a gram-positive and thermophilic bacterium, and cloned the gene encoding MQO based on the obtained partial N-terminus sequence. Purified MQOs showed a molecular mass of ~90 kDa, which was estimated using gel filtration, and it consists of two subunits with a molecular mass of ~50 kDa. Phylogenetic analysis showed a high similarity to the MQO of the Geobacillus group rather than the Bacillus group. Additionally, the purified enzyme was thermostable and it retained menaquinol reduction activity at high temperatures. Although it is difficult to conduct experiments using menaquinol because of its instability, we were able to measure the oxidase activity of cytochrome bd-type quinol oxidase by using menaquinol-1 by coupling this molecule with the menaquinol reduction reaction using purified MQOs.

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

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 b 595 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.

Monitoring enzyme expression of a branched respiratory chain of corynebacterium glutamicum using an EGFP reporter gene.

To investigate the expressional control of branched respiratory chain complexes of the amino-acid producing bacterium Corynebacterium glutamicum according to growth conditions, the expression indexes of the ndh, sdh, qcrCAB, ctaCF, ctaD, ctaE, and cydAB genes were estimated under aerobic and microaerobic, and carbon-rich and -poor conditions. The promoter region of each target gene was cloned upstream of the EGFP gene on expression vector pVK6, and the nine reporter constructs were transformed into C. glutamicum ssp. lactofermentum. The cytochrome content of cellular membranes obtained from each growth phase closely corresponded to the expression indexes based on EGFP fluorescence and cell density, indicating that this rapid and convenient method is suitable for analyzing the expression levels of respiratory chain complexes. Using this method, we demonstrated that a reciprocal change in the expression levels of cytochrome bd-type and aa (3)-type oxidases occurs when C. glutamicum cells are held in stationary phase for extended periods.

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

BACKGROUND: The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species. RESULTS: Here, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectrum of the isolated c-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 c subunit encoded by the ORF APE_1719.1. In addition, the c-type cytochrome-containing polypeptide complex exhibited menaquinone: yeast cytochrome c oxidoreductase activities. CONCLUSION: In conclusion, we showed that A. pernix, a hyperthemophilic archaeon, has a "full" bc complex that includes a c-type cytochrome, and to the best of our knowledge, A. pernix is the first archaea from which such a bc complex has been identified. However, an electron donor candidates for cytochrome c 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 bc complex and terminal 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-aa(3)-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.

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 bo(3)-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 bo(3), 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 k(cat) value most prominently without affecting the K(m) value, indicating that the residue is important for electron transfer. The mutation of D99, located close to the Cu(A) 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 bo(3) for cytochrome c-551 without affecting the k(cat) value. These three residues are located at the front of the hydrophilic globular domain and distant from the Cu(A) 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.

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 aa(3)-type cytochrome c oxidase and bd-type menaquinol oxidase. Thus, the chain has two branches of electron flow. The bcc-aa(3) 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 (DeltactaD) or subunits I and II of the quinol oxidase (DeltacydAB), 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 DeltactaD mutant. In contrast, the value was 5.23 +/- 0.36 for the DeltacydAB mutant. The cells grown in the LB medium showed higher value compared to that of cells grown in the semi-synthetic medium. The DeltactaD 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.