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.

First evidence for the presence of a hydrogenase in the sulfur-reducing bacterium Desulfuromonas acetoxidans.

Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from Desulfuromonas acetoxidans. For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium D. acetoxidans, grown on ethanol-malate, was detected and enriched. To assay the role of the hydrogenase in the energetic metabolism of D. acetoxidans, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium.

Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent phosphorylation.

Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J. (1994) J. Biol. Chem. 269, 26559-26567). Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower Km value for the co-substrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMP-dependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser32, a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects.

15N-labelling and preliminary heteronuclear NMR study of Desulfovibrio vulgaris Hildenborough cytochrome c553.

When using heteronuclear NMR, 15N-labelling is necessary for structural analysis, dynamic studies and determination of complex formation. The problems that arise with isotopic labelling of metalloproteins are due to their complex maturation process, which involves a large number of factors. Cytochromes c are poorly expressed in Escherichia coli and the overexpression that is necessary for 15N-labelling, requires an investigation of the expression host and special attention to growth conditions. We have succeeded in the heterologous expression and the complete and uniform isotopic 15N-labelling of the cytochrome c553 from Desulfovibrio vulgaris Hildenborough, in a sulphate-reducing bacterium, D. desulfuricans G200, by using a growth medium combining 15N-ammonium chloride and 15N-Celtone. These conditions allowed us to obtain approximately 0.8 mg x L-1 of pure labelled cytochrome c553. 1H and 15N-assignments for both the oxidized and the reduced states of cytochrome c553 were obtained from two-dimensional heteronuclear experiments. Pseudocontact effects due to the haem Fe3+ have been analysed for the first time through 15N and 1H chemical shifts in a c-type cytochrome.

In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri.

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.

Influence of "energization" on the binding of M protein with p-nitrophenyl alpha-D-galactopyranoside.

A specific binding of p-nitrophenyl alpha-D-galactopyranoside has been measured by flow dialysis with Escherichia coli ML 308225 membrane vesicles containing the lac carrier protein. The number of binding sites, 0.45 nmol/mg of membrane protein, remains unchanged in the presence or absence of energy. On the other hand, "energization" increases the M protein affinity for p-nitrophenyl alpha-D-galactopyranoside. The dissociation constant (Kd) is 4 and 21 microM in the presence and absence, respectively, of D-lactate. The same energization effects are found with E. coli A3245 membrane vesicles. p-Nitrophenyl alpha-D-galactopyranoside can be used as a substrate to study energization effect on binding to the lactose permease M protein. These results corroborate observations that energy increases the lac carrier protein affinity for its substrate, and they also confirm the concentration of the M protein, which corresponds to 1.4% of the membrane protein.