Solution structure of the functional domain of Paracoccus denitrificans cytochrome c₅₅₂ in the reduced state

Abstract

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...

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