Expression of cytochrome c oxidase subunit 1 gene in the brain at an early stage in the termination of pupal diapause in the sweet potato hornworm, Agrius convolvuli

Suppression-subtractive hybridization was used to isolate cDNAs specifically expressed in the brain at the termination of pupal diapause in Agriusconvolvuli. One of the isolated clones shows similarity to the cytochrome c oxidase subunit 1 (COX1) gene. The full-length cDNA was obtained from brain mRNA by rapid amplification of cDNA ends (RACE). The insert is 1.65 kb in length and has an open reading frame of 1.46 kb which encodes a putative protein of 486 amino acid residues. RT-PCR reveals that the mRNA increases dramatically at an early stage of diapause termination. Activity of cytochrome c oxidase in the brain also increases at the same time. The up-regulation of this gene suggests that expression of the COX1 gene and ATP synthesis are initiated in the brain in association with diapause termination.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans

Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba₃-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa₃-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa₃-type cytochrome c oxidase and the ba₃-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.     

The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo–formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: II. KINETICS AND MECHANISM OF BINDING*

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c₁ of detergent-solubilized bc₁ complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c₁-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mm Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mm, the rate leveled off, indicating a rate-limiting conformational step with lifetime ∼1 s; and (iii) at Im concentrations above 100 mm, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.     

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF₁F_O-ATP synthase) of plants is integrated into the thylakoid membrane via its F_O-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H⁺-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F₁ sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF₁F_O-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.     

c-Type Cytochrome Biogenesis Can Occur via a Natural Ccm System Lacking CcmH,CcmG, and the Heme-binding Histidine of CcmE

The Ccm cytochrome c maturation System I catalyzes covalent attachment of heme to apocytochromes c in many bacterial species and some mitochondria. A covalent, but transient, bond between heme and a conserved histidine in CcmE along with an interaction between CcmH and the apocytochrome have been previously indicated as core aspects of the Ccm system. Here, we show that in the Ccm system from Desulfovibrio desulfuricans, no CcmH is required, and the holo-CcmE covalent bond occurs via a cysteine residue. These observations call for reconsideration of the accepted models of System I-mediated c-type cytochrome biogenesis.     

Nrg1 functions as a global transcriptional repressor of glucose-repressed genes through its direct binding to the specific promoter regions

Nrg1 is a zinc finger protein involved in the glucose repression of several glucose-repressed genes such as STA1, SUC2, and GAL1. Although the molecular details of the Nrg1-mediated repression of STA1 have been partly characterized, it still remains largely unknown how Nrg1 regulates these multiple target genes. In this study, we show that Nrg1 mediates the glucose repression of SUC2 and HXT2 through its direct binding to the specific promoter regions; it binds to the −404 to −360 region of the SUC2 promoter and the −957 to −810 region of the HXT2 promoter. Nrg1 also interacts with the −380 to −250 region of the PCK1 promoter, suggesting that it might also contribute to the PCK1 repression. In addition, ChIP assays confirmed that Nrg1 associated with specific promoter regions of these glucose-repressed genes in vivo. Analysis of the DNA fragments to which it binds indicates that Nrg1 may recognize T/ACCCC sequence within the promoters of these glucose-repressed genes as well as in its own promoter. Collectively, our findings indicate that Nrg1 mediates the glucose repression of multiple genes through its direct binding to the specific promoter regions.