RESUMO
The helix-turn-helix (HTH) motif features frequently in protein DNA-binding assemblies. Viral pac site-targeting small terminase proteins possess an unusual architecture in which the HTH motifs are displayed in a ring, distinct from the classical HTH dimer. Here we investigate how such a circular array of HTH motifs enables specific recognition of the viral genome for initiation of DNA packaging during virus assembly. We found, by surface plasmon resonance and analytical ultracentrifugation, that individual HTH motifs of the Bacillus phage SF6 small terminase bind the packaging regions of SF6 and related SPP1 genome weakly, with little local sequence specificity. Nuclear magnetic resonance chemical shift perturbation studies with an arbitrary single-site substrate suggest that the HTH motif contacts DNA similarly to how certain HTH proteins contact DNA non-specifically. Our observations support a model where specificity is generated through conformational selection of an intrinsically bent DNA segment by a ring of HTHs which bind weakly but cooperatively. Such a system would enable viral gene regulation and control of the viral life cycle, with a minimal genome, conferring a major evolutionary advantage for SPP1-like viruses.
Assuntos
Fagos Bacilares/genética , Endodesoxirribonucleases/metabolismo , Montagem de Vírus/fisiologia , Fagos Bacilares/fisiologia , Sítios de Ligação , DNA/química , DNA/metabolismo , Empacotamento do DNA , DNA Viral/química , DNA Viral/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Sequências Hélice-Volta-Hélice , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Ultracentrifugação/métodos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus/genéticaRESUMO
NADH-OH, the specific inhibitor of NADH-binding site of the mammalian complex I, is shown to completely block FMN-dependent reactions of P. denitrificans enzyme in plasma membrane vesicles: NADH oxidation (in a competitive manner with Ki of 1 nM) as well as reduction of pyridine nucleotides, ferricyanide and oxygen in the reverse electron transfer. In contrast to these activities, the reverse electron transfer to hexaammineruthenium (III) catalyzed by plasma membrane vesicles is insensitive to NADH-OH. To explain these results, we hypothesize the existence of a non-FMN redox group of P. denitrificans complex I that is capable of reducing hexaammineruthenium (III), which is corroborated by the complex kinetics of NADH: hexaammineruthenium (III)-reductase activity, catalyzed by this enzyme. A new assay procedure for measuring succinate-driven reverse electron transfer catalyzed by P. denitrificans complex I to hexaammineruthenium (III) is proposed.
Assuntos
NAD , Paracoccus denitrificans , Animais , NAD/metabolismo , Ubiquinona/metabolismo , Prótons , Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Membrana Celular/metabolismo , Mamíferos/metabolismoRESUMO
Mitochondrial NADH:ubiquinone oxidoreductase (proton translocating respiratory complex I) serves several essential functions in cell metabolism: it maintains the intramitochondrial NADH/NAD+ ratio, contributes to generation of the proton-motive force, and participates in physiological and/or pathophysiological production of so-called reactive oxygen species. A characteristic feature of complex I is a slow, compared with its catalytic turnover, transformation to its inactive (deactivated) state, a phenomenon operationally called A/D transition. Here we report data on several extrinsic factors affecting deactivation as observed in coupled or uncoupled bovine heart submitochondrial particles. The time course of the strongly temperature-dependent deactivation deviates from first-order kinetics, and this deviation is abolished in the presence of an SH-group-specific reagent. The residual fraction of activity attained upon extensive deactivation shows the same kinetics of NADH oxidation as the fully active enzyme does. The rate of complex I deactivation is only slightly pH dependent within the range of 7.0-8.5 and significantly increases at higher pH. ATPâ(Mg) decreases the rate of complex I deactivation in coupled SMP, and this effect is abolished if the proton-motive force generating ATPase activity of FoâF1 is precluded. Taken together, the data show that an equilibrium between the A and D forms of complex I exists. Possible mechanistic aspects of the deactivation process are discussed.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Animais , Bovinos , Membrana Celular/metabolismo , Complexo I de Transporte de Elétrons/química , Ativação Enzimática , Cinética , Desdobramento de Proteína , PrótonsRESUMO
A simple assay procedure for measuring ATP-dependent reverse electron transfer from ubiquinol to hexaammineruthenium (III) (HAR) catalyzed by mitochondrial respiratory complex I is introduced. The specific activity of the enzyme in this reaction and its sensitivity to the standard inhibitors and uncoupling are the same as with other well-known electron acceptors, NAD+ and ferricyanide. In contrast to the reactions with these acceptors, the energy-dependent HAR reduction is not inhibited by NADH-OH, the specific inhibitor of NADH-binding site. These results suggest that a catalytically competent electron connection exists between HAR and a redox component of complex I that is different from flavin mononucleotide bound at the substrate-binding site.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Transporte de Elétrons/fisiologia , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Animais , Sítios de Ligação , Bovinos , Complexo I de Transporte de Elétrons/química , Elétrons , Transferência de Energia/fisiologia , Técnicas In Vitro , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Oxirredução , Compostos de Rutênio/química , Compostos de Rutênio/farmacologiaRESUMO
The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.