The vertebrate homologue of sulfide-quinone reductase in mammalian mitochondria.

Hydrogen sulfide (H2S) is the first inorganic compound identified as both a substrate for mitochondrial oxidative phosphorylation and a transmitter in mammalian cells. H2S seems to mediate effects that are correlated with those of nitric oxide (NO) by a reciprocal regulation. Moreover, H2S is consumed by mitochondrial oxidation mediated by sulfide-quinone reductase-like protein (SQRDL)-the vertebrate homolog of sulfide-quinone oxidoreductase (SQR). There is evidence that SQR plays an essential role in regulating H2S levels in fission yeast. To start understanding the role of SQRDL in the mammalian metabolism of H2S, we examine rat tissues. Our results show that SQRDL protein is present in all tissues tested, albeit restricted to specific mitochondrial populations at the cellular level. We demonstrate a developmental regulation of Sqrdl transcription in the kidney, where SQRDL protein is detectable in glomerular podocytes and in tubular cells of the renal medulla. We also show that Sqrdl transcription in T cells is responsive to external H2S. Taken together, our results suggest that Sqrdl transcription is adaptively regulated, probably to meet the need of H2S oxidation. Thus far, SQRDL has only been studied in a limited set of tissues. The present report demonstrates the presence and specific localization of SQRDL in various mammalian tissues.

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation.

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain. Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.

Vectorial redox reactions of physiological quinones. I. Requirement of a minimum length of the isoprenoid side chain.

Physiological quinones carrying isoprenoid side chains have been compared with homologues lacking the side chain, for their ability to carry electrons and protons from dithionite to ferricyanide, trapped in liposomes. Six differential observations were made: (1) Plastoquinone and ubiquinones, with a side chain of more than two isoprene units, are by far better mediators than their short-chain homologues. Also other benzoquinones lacking a long side chain are poor catalysts, except dimethyl-methylenedioxy-p-benzoquinone, a highly autooxidizable compound. Tocopherol is a good catalyst. (2) Vitamin K-1 and K-2 are poor mediators compared to vitamin K-3. (3) The reaction catalyzed by quinones carrying long isoprenoid side chains has an about three-fold higher activation energy, irrespective of the catalytic efficiency. (4) The reaction catalyzed by quinones lacking a long side chain follows pseudo first-order kinetics, while the reaction with quinones carrying a long side chain is of apparently higher order. (5) The rate with ubiquinone-1 is increasing pH, while with ubiquinone-9 it is decreasing. (6) The reaction mediated by short-chain quinones seems to be satuarated at lower dithionite concentration. We conclude that isoprenoid quinones are able to translocate electrons and protons in lipid membranes, and that the side chain has a strong impact on the mechanism. This and the relevance of the model reaction for electron and proton transport in photosynthesis and respiration is discussed.

Cyanidium caldarium genes encoding subunits A and B of V-ATPase.

The genes encoding subunits A and B of V-ATPase in Cyanidium caldarium were cloned and sequenced. While the gene encoding subunit A is not interrupted by introns, the gene encoding subunit B contains seven introns ranging from 36 to 60 nucleotides.

The reaction center of green sulfur bacteria(1).

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.

The photosystem I-like P840-reaction center of green S-bacteria is a homodimer.

An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies.

Spin polarized transient EPR spectra taken at X-band (9 GHz) and K-band (24 GHz) of membrane fragments of Chlorobium tepidum and Heliobacillus mobilis are presented along with the spectra of two fractions obtained in the purification of reaction centers (RC) from C. tepidum. The lifetime of P+. is determined by measuring the decay of the EPR signals following relaxation of the initial spin polarization. All samples except one of the RC fractions show evidence of light induced charge separation and formation of chlorophyll triplet states. The lifetime of P+. is found to be biexponential with components of 1.5 ms and 30 ms for C. tepidum and 1.0 and 4.5 ms for Hc. mobilis at 100 K. In both cases, the rates are assigned to recombination from F-X. The spin polarized radical pair spectra for both species are similar and those from Hc. mobilis at room temperature and 100 K are identical. In all cases, an emission/absorption polarization pattern with a net absorption is observed. A slight narrowing of the spectra and a larger absorptive net polarization is found at K-band. No out-of-phase echo modulation is observed. Taken together, the recombination kinetics, the frequency dependence of the spin polarization and the absence of an out-of-phase echo signal lead to the assignment of the spectra to the contribution from P+. to the state P+.F-X. The origin of the net polarization and its frequency dependence are discussed in terms of singlet-triplet mixing in the precursor. It is shown that the field-dependent polarization expected to develop during the 600-700 ps lifetime of P+.A-.0 is in qualitative agreement with the observed spectra. The identity that the acceptor preceding FX and the conflicting evidence from EPR, optical methods and chemical analyses of the samples are discussed.

Asymmetry of an energy transducing membrane the location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata.

Monospecific antibodies have been prepared against cytochrome c2 from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c' from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth. Cytochrome c2 is located in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.

The atp2 operon of the green bacterium Chlorobium limicola.

The operon (atp2) encoding the beta and epsilon subunits of F-ATPase from Chlorobium limicola was cloned and sequenced. In contrast with purple bacteria these genes are arranged in a separate operon similar to the cyanobacteria. The operon terminates with a pronounced stem-loop structure. About 0.8 kb upstream of the beta subunit a gene encoding the enzyme phospho enol pyruvate carboxykinase was identified. This gene is transcribed in the opposite direction of the atp2 operon and also ends with a stem-loop structure. These genes of green bacteria are among the first to be sequenced, and therefore the genetic distance between these genes and corresponding genes from other bacteria and eukaryotes was studied. Even though the operon structure resembles that of cyanobacteria, the evolutionary tree compiled from these data places the chlorobium gene close to purple bacteria. Chlorobium limicola beta and epsilon subunits complemented Escherichia coli mutants defective in the corresponding subunits, indicating that the hybrid enzyme formed from subunits of the two bacteria is active in ATP synthesis.

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.

Early evolution of cytochrome bc complexes.

Primary structures, functional characteristics and phylogenetic relationships of subunits of cytochrome bc complexes from phylogenetically diverse bacterial and archaeal species were analysed. A single case of lateral gene transfer, i.e. the import of an epsilon-proteobacterial cytochrome bc(1) complex into Aquificales, was identified. For the enzyme in the remainder of the species studied, the obtained phylogenies were globally in line with small subunit rRNA trees. The distribution of a few key phylogenetic markers, such as contiguousness of cytochrome b, nature of the c-type subunit or spacing between b-heme ligands, are discussed. A localised modification of previous tree topologies is proposed on the basis of the obtained data. The comparison of extant enzymes furthermore allowed us to define the minimal functional and evolutionary core of the enzyme. The data furthermore suggest that the ancestral enzyme was put together from subunits that previously had played a role in other electron transfer chains.

Characterization of cytochrome b in the isolated ubiquinol-cytochrome c2 oxidoreductase from Rhodopseudomonas sphaeroides GA.

Extinction coefficients for cytochrome b and c1 in the isolated cytochrome bc1 complex from Rhodopseudomonas sphaeroides GA have been determined. They are 25 mM-1 . cm-1 at 561 nm for cytochrome b and 17.4 mM-1 . cm-1 at 553 nM for cytochrome c1, for the difference between the reduced and the oxidized state. Cytochrome b is present in two forms in the complex. One form has an Em7 of 50 mV, an alpha-peak of 557 nm at liquid N2 temperature and of 561 nm at RT, which is red-shifted by antimycin A. The other form has an Em7 of -90 mV, a double alpha-peak of 555 and 561 nm at liquid N2 temperature corresponding to 559 and 566 nm at RT. The absorption at 566 nm is red-shifted by myxothiazol. The two shifts are independent of each other. Both midpoint potentials of cytochromes b are pH-dependent. The redox center compositions of the cytochrome bc1 complexes from Rhodopseudomonas sphaeroides and from mitochondria are identical.

Sulfide quinone reductase (SQR) activity in Chlorobium.

Membranes of the green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum, catalyze the reduction of externally added isoprenoid quinones by sulfide. This activity is highly sensitive to stigmatellin and aurachins. It is also inhibited by 2-n-nonyl-4-hydroxyquinoline-N-oxide, antimycin, myxothiazol and cyanide. It is concluded that in sulfide oxidizing bacteria like Chlorobium, sulfide oxidation involves a sulfide-quinone reductase (SQR) similar to the one found in Oscilatoria limnetica [Arieli, B., Padan, E. and Shahak, Y. (1991) J. Biol. Chem. 266, 104-111].

ATP synthesis at 100 degrees C by an ATPase purified from the hyperthermophilic archaeon Pyrodictium abyssi.

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100 degrees C and gains energy by sulfur respiration, with hydrogen as electron donor. From the membranes of this hyperthermophile, an ATPase complex was isolated. The purified enzyme consists of six major polypeptides, the 67, 51, 41, 26 and 22 kDa subunits composing the AF(1) headpiece, and the 7 kDa proteolipid of the AF(0) component. The headpiece of the enzyme restored the formation of ATP during sulfur respiration in membrane vesicles from which it had been removed by low salt treatment. Characteristics of the reconstituted activity suggest that the same enzyme is responsible for ATP formation in untreated membranes. ATP formation was neither sensitive to ionophores and uncouplers, nor to dicyclohexyl carbodiimide, but depended on closed vesicles. Both ATPase activity (up to 2 micromol per min and mg protein) as well as ATP formation (up to 0.4 micromol per min and mg membrane protein) were highest at 100 degrees C. A P/e2 ratio of close to one can be estimated for sulfur respiration with hydrogen. In addition to ATP, autoradiographic detection revealed the formation of high quantities of (33)P(i)-labeled ADP and of another compound not identified so far.

The vertebrate homolog of sulfide-quinone reductase is expressed in mitochondria of neuronal tissues.

Hydrogen sulfide (H2S) can be consumed by both invertebrates and vertebrates as an inorganic substrate. The pathway metabolizing H2S probably involves three mitochondrial enzymes, one of which is sulfide-quinone oxidoreductase (SQR), known as sulfide-quinone reductase-like protein (SQRDL) in vertebrates. Evidence from fission yeast suggests that SQR might have a role in regulating sulfide levels in the cell. Regulation might be essential for H2S to act as a gaseous transmitter (gasotransmitter). The brain is an organ with high activity of gasotransmitters, like nitric oxide (NO) and H2S, which are known to affect synaptic transmission. In this study, we provide evidence that SQRDL is expressed in the mammalian brain. Real-time polymerase chain reaction (PCR) showed an increase in the number of Sqrdl transcripts in the brain with increasing age. Cellular fractionation and subsequent analysis by Western blotting indicated that the protein is located in mitochondria, which is the site of sulfide consumption in the cell. With an immunohistochemical approach, we demonstrated that the SQRDL protein is expressed in neurons, oligodendrocytes, and endothelial cells. Taken together, our data suggest that brain tissue harbors the machinery required for local regulation of sulfide levels.