No abstract

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

A soluble and fully functional 10.5 kDa fragment of the 18.2 kDa membrane-bound cytochrome c(552) from Paracoccus denitrificans has been heterologously expressed and (13)C/(15)N-labeled to study the structural features of this protein in both redox states. Well-resolved solution structures of both the reduced and oxidized states have been determined using high-resolution heteronuclear NMR. The overall protein topology consists of two long terminal helices and three shorter helices surrounding the heme moiety. No significant redox-induced structural differences have been observed. (15)N relaxation rates and heteronuclear NOE values were determined at 500 and 600 MHz. Several residues located around the heme moiety display increased backbone mobility in both oxidation states, while helices I, III, and V as well as the two concatenated beta-turns between Leu30 and Arg36 apparently form a less flexible domain within the protein structure. Major redox-state-dependent differences of the internal backbone mobility on the picosecond-nanosecond time scale were not evident. Hydrogen exchange experiments demonstrated that the slow-exchanging amide proton resonances mainly belong to the helices and beta-turns, corresponding to the regions with high order parameters in the dynamics data. Despite this correlation, the backbone amide protons of the oxidized cytochrome c(552) exchange considerably faster with the solvent compared to the reduced protein. Using both differential scanning calorimetry as well as temperature-dependent NMR spectroscopy, a significant difference in the thermostabilities of the two redox states has been observed, with transition temperatures of 349.9 K (76.8 degrees C) for reduced and 307.5 K (34.4 degrees C) for oxidized cytochrome c(552). These results suggest a clearly distinct backbone stability between the two oxidation states.

In order to determine the solution structure of Paracoccus denitrificans cytochrome c 552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15 N-enriched samples of cytochrome c 552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1 H and 15 N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56^0.10 A Ê and 1.09^0.09 A Ê for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups.Keywords: cytochrome c 552 ; heme; isotope enrichment { 15 N}; NMR spectroscopy; redox states.Cytochrome c is a well-established electron mediator between the last two complexes of the mitochondrial redox chain, the cytochrome bc 1 complex (complex III) and the aa 3 -type cytochrome c oxidase (complex IV). It contains a covalently bound heme moiety, with thioether linkages to the cysteine residues in the conserved CXYCH motif. During the redox cycle, the iron atom alternates between the diamagnetic reduced (21) and the paramagnetic oxidized (31) state. It is octahedrally coordinated by the four porphyrin nitrogens and two axial ligands, a histidine and a methionine, which is a common feature in this class of cytochromes [1,2].Respiratory electron-transfer (ET) complexes are integral membrane proteins, which are not easily amenable to structural studies. The molecular basis of ET has therefore been understood in detail for only a few selected model systems [3,4]. The use of heteronuclear NMR is critically dependent on efficient isotope labeling of the protein, often only achieved in heterologous expression hosts such as Escherichia coli, in which the biosynthesis of the heme cofactor, its translocation across the cytoplasmic membrane, and the insertion into the apo-protein pose an additional challenge. Thus, the difficulty in labeling cytochromes is due to the complexity of the protein maturation. Until now, to the best of our knowledge, 13 C/ 15 N-labeling has been reported only for R. capsulatus cytochrome c 2 [23], cytochrome c H from purple bacteria [24] and Thiobacillus versutus ferrocytochrome c 550 [25], as well as Greater nutritional adaptability and flexibility to environmental demands often cause bacterial ET chains to display a higher degree o...

Under in vitro conditions, bacterial cytochrome c oxidases may accept several nonhomologous c-type electron donors, including the evolutionarily related mitochondrial cytochrome c. Several lines of evidence suggest that in intact membranes the heme aa(3) oxidase from Paracoccus denitrificans receives its electrons from the membrane-bound cytochrome c(552). Both the structures of the oxidase and of a heterologously expressed, soluble fragment of the c(552) have been determined recently, but no direct structural information about a static cocomplex is available. Here, we analyze the kinetic properties of the isolated oxidase with the full-size c(552), with two truncated soluble forms, and with a set of site-specific mutants within the presumed docking site of the cytochrome, all heterologously expressed in Escherichia coli. Our data indicate that all three forms, the wild type and both truncations, are fully competent kinetically and exhibit biphasic kinetic behavior, however, under widely different ionic strength conditions. When mutations in lysine residues clustered around the interaction domain were introduced into the smallest fragment of c(552), both kinetic parameters, K(M) and k(cat), were drastically influenced. On the other hand, when the nonmutated truncated form was used to donate electrons to a set of oxidase mutants with replacements clustered along the docking site on subunit II, we observe distinct differences when comparing the kinetic properties of the widely used horse heart cytochrome c with those of the bacterial c(552). We conclude that the specific docking sites for the two types of cytochromes differ to some extent.