petR, located upstream of the fbcFBC operon encoding the cytochrome bc1 complex, is homologous to bacterial response regulators and necessary for photosynthetic and respiratory growth of Rhodobacter capsulatus

Interposon mutagenesis of a region upstream of the petABC(fbcFBC) operon, encoding the ubiquinol: cytochrome c2 oxidoreductase (bc1 complex) of the photosynthetic bacterium Rhodobacter capsulatus revealed the presence of two genes, petP and petR. DNA nucleotide sequence determination of this region indicated that petP and petR are transcribed in the same direction as the petABC(fbcFBC) operon, and are translationally coupled. A silent insertion located in the interoperonal region separating petPR and the petABC(fbcFBC) genes indicated that these clusters have separate promoters. The deduced amino acid sequence of the putative petR gene product is homologous to various bacterial response regulators, especially to those of the OmpR subgroup. Moreover, it was found that PetR mutants are unable to grow on rich or minimal media by either photosynthesis or respiration, demonstrating that these gene products are essential for growth of R. capsulatus.     

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

Cytochrome c maturation (Ccm) is a post-translational process that occurs after translocation of apocytochromes c to the positive (p) side of energy-transducing membranes. Ccm is responsible for the formation of covalent bonds between the thiol groups of two cysteines residues at the heme-binding sites of the apocytochromes and the vinyl groups of heme b (protoporphyrin IX-Fe). Among the proteins (CcmABCDEFGHI and CcdA) required for this process, CcmABCD are involved in loading heme b to apoCcmE. The holoCcmE thus formed provides heme b to the apocytochromes. Catalysis of the thioether bonds between the apocytochromes c and heme b is mediated by the heme ligation core complex, which in Rhodobacter capsulatus contains at least the CcmF, CcmH, and CcmI components. In this work we show that the heme chaperone apoCcmE binds to the apocytochrome c and the apocytochrome c chaperone CcmI to yield stable binary and ternary complexes in the absence of heme in vitro. We found that during these protein-protein interactions, apoCcmE favors the presence of a disulfide bond at the apocytochrome c heme-binding site. We also establish using detergent-dispersed membranes that apoCcmE interacts directly with CcmI and CcmH of the heme ligation core complex CcmFHI. Implications of these findings are discussed with respect to heme transfer from CcmE to the apocytochromes c during heme ligation assisted by the core complex CcmFHI.     

The Cytochrome bc 1 Complex and its Homologue the b 6 f Complex: Similarities and Differences

The ubihydroquinone:cytochrome c oxidoreductase (also called complex III, or bc (1) complex), is a multi subunit enzyme encountered in a very broad variety of organisms including uni- and multi-cellular eukaryotes, plants (in their mitochondria) and bacteria. Most bacteria and mitochondria harbor various forms of the bc (1) complex, while plant and algal chloroplasts as well as cyanobacteria contain a homologous protein complex called plastohydroquinone:plastocyanin oxidoreductase or b (6) f complex. Together, these enzyme complexes constitute the superfamily of the bc complexes. Depending on the physiology of the organisms, they often play critical roles in respiratory and photosynthetic electron transfer events, and always contribute to the generation of the proton motive force subsequently used by the ATP synthase. Primarily, this review is focused on comparing the 'mitochondrial-type' bc (1) complex and the 'chloroplast-type' b (6) f complex both in terms of structure and function. Specifically, subunit composition, cofactor content and assembly, inhibitor sensitivity, proton pumping, concerted electron transfer and Fe-S subunit large-scale domain movement of these complexes are discussed. This is a timely undertaking in light of the structural information that is emerging for the b (6) f complex.     

Acylated flavone glycosides from Veronica

A survey of the flavonoid glycosides of selected taxa in the genus Veronica yielded two new acylated 5,6,7,3',4'-pentahydroxyflavone (6-hydroxyluteolin) glycosides and two unusual allose-containing acylated 5,7,8,4'-tetrahydroxyflavone (isoscutellarein) glycosides. The new compounds were isolated from V. liwanensis and V. longifolia and identified using NMR spectroscopy as 6-hydroxyluteolin 4'-methyl ether 7-O-alpha-rhamnopyranosyl(1"'-->2")[6"-O-acetyl-beta-glucopyranoside] and 6-hydroxyluteolin 7-O-(6"-O-(E)-caffeoyl)-beta-glucopyranoside, respectively. Isoscutellarein 7-O-(6"'-O-acetyl)-beta-allopyranosyl(1"'-->2")-beta-glucopyranoside was obtained from both V. intercedens and V. orientalis and its 4'-methyl ether from V. orientalis only. Complete 1H and 13C NMR spectral assignments are presented for both isoscutellarein glycosides. Two iridoid glucosides new to the genus Veronica (melittoside and globularifolin) were also isolated from V. intercedens.     

Membrane-anchored cytochrome c as an electron carrier in photosynthesis and respiration: past,present and future of an unexpected discovery

In the mid 1980s, it was observed that photosynthesis could still occur in the absence of the diffusible electron carrier cytochrome c ₂ in the purple non-sulfur facultative phototrophic bacterium Rhodobacter capsulatus. This serendipic finding led to the discovery of a novel class of membrane-anchored electron carrier cytochromes and their associated electron transfer pathways. Studies of cytochrome c _y of R. capsulatus (and its homologues in other species) have modified the previous dogma of electron transfer between photosynthetic and respiratory membrane protein complexes with a new paradigm, in which these proteins and their electron carriers can form `hard-wired' structural super-complexes. Here, we reminisce on the early days of this discovery, its impacts on our understanding of cellular energy transduction pathways and the physiological roles played by the electron carrier cytochromes c, and discuss the current knowledge and emerging future challenges of this field. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytochrome c biogenesis System I: An intricate process catalyzed by a maturase supercomplex?

Cytochromes c are ubiquitous heme proteins that are found in most living organisms and are essential for various energy production pathways as well as other cellular processes. Their biosynthesis relies on a complex post-translational process, called cytochrome c biogenesis, responsible for the formation of stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of apocytochromes c heme-binding site (C₁XXC₂H) cysteine residues. In some organisms this process involves up to nine (CcmABCDEFGHI) membrane proteins working together to achieve heme ligation, designated the Cytochrome c maturation (Ccm)-System I. Here, we review recent findings related to the Ccm-System I found in bacteria, archaea and plant mitochondria, with an emphasis on protein interactions between the Ccm components and their substrates (apocytochrome c and heme). We discuss the possibility that the Ccm proteins may form a multi subunit supercomplex (dubbed “Ccm machine”), and based on the currently available data, we present an updated version of a mechanistic model for Ccm. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Genetic Structure of Brown Trout (Salmo trutta) Populations from Turkey Based on Microsatellite Data

This present study investigated micro- and macro-geographic microsatellite DNA variations using five polymorphic microsatellite loci from 27 brown trout populations in Turkey. Average number of alleles and average observed heterozygosity were 7.4 and 0.254, respectively. Even populations from the same sea basin and river system (the so called micro-geographic regions) had unique alleles. Genetic variation among the populations from macro-geographic regions (different sea basins and river systems) was 45.78%. The mtDNA lineages of brown trout that have previously been identified by mtDNA analyses were supported by the analysis of the microsatellite DNA data in general. The Çatak population, which belongs to the Tigris lineage, was clustered together with the Euphrates populations within the Adriatic mtDNA lineage, based on microsatellite data. Both mitochondrial and microsatellite DNA analyses have made it possible to determine a secondary contact between Adriatic and Tigris lineages.     

Triterpene glycosides from Astragalus icmadophilus

Six cycloartane-type triterpene glycosides were isolated from Astragalus icmadophilus along with two known cycloartane-type glycosides, five known oleanane-type triterpene glycosides and one known flavonol glycoside. The structures of the six compounds were established as 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxy cycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3,4-diacetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane by the extensive use of 1D- and 2D-NMR experiments along with ESIMS and HRMS analysis.The first four compounds are cyclocanthogenin and cycloastragenol glycosides, whereas the last two are based on cyclocephalogenin as aglycone, more unusual in the plant kingdom, so far reported only from Astragalus spp.     

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c₂ by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c₂ and c₁, and class II c′) and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nm to μm K_D values). When present, heme induced conformational changes in class I apocytochromes (e.g. c₂) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c₂ and that heme converts apocytochrome c₂ into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c₂) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.     

Binding dynamics at the quinone reduction (Qi) site influence the equilibrium interactions of the iron sulfur protein and hydroquinone oxidation (Qo) site of the cytochrome bc1 complex

Multiple instances of low-potential electron-transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc(1) complex to varying degrees, ranging from changes in hydroquinone (QH(2)) oxidation and cyt c(1) reduction kinetics to proteolytic accessibility of the hinge region of the iron-sulfur-containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation-dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low-potential electron-transport pathway by Q(i)-site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Q(i)-site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster and a second affecting the mobility of this subunit, are revealed. Remarkably, different inhibitors and mutations at or near the Q(i) site induce these two effects differently, indicating that the events occurring at the Q(i) site affect the global structure of the cyt bc(1). Furthermore, occupancy of discrete Q(i)-site subdomains differently impede the location of the Fe/S protein at the Q(o) site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH(2) and Q at the Q(i) site, respectively. Implications of these findings in respect to the Q(o)-Q(i) sites communications and to multiple turnovers of the cyt bc(1) are discussed.