Structural and biochemical characterization of diheme <i>c</i>-type cytochromes

Abstract

Cytochromes are an abundant family of proteins incorporating heme as a prosthetic group and are involved in processes of electron conduction or redox reactions. Heme consists of an aromatic porphyrin macrocycle and the central coordinated iron ligand. Cytochromes of the c-type bind the heme group covalently to the protein backbone. A conserved amino acid heme binding motif is therefore providing two cysteine sulfhydryl groups, to which the heme group is attached via two thioether bonds. The covalent attachment allows for unique properties, such as massive heme group clustering within a protein. X-ray crystallography experiments revealed the presence of a perpendicular and a parallel structural conserved heme group packing motif. Investigations on these motifs are often complicated by the sheer number of heme groups in multi heme cytochromes, as biochemical and spectroscopic data is summing up and therefore indistinguishable.The recombinant diheme c-type cytochrome DHC2 from the anaerobic microorganism Geobacter sulfurreducens was overexpressed in Escherichia coli and purified to homogeneity using affinity- and gelfiltration chromatography. The protein was characterized using UV/Vis absorption spectroscopy, electron paramagnetic resonance spectroscopy and redox titrations. DHC2 was crystallized and its structure solved by an X-ray diffraction experiment.The structure is showing two monomers in the asymmetric unit, as well as two covalently bound heme groups per monomer, showing a structural conserved heme packing motif. The structural arrangement of amino acids at the heme groups is reflected by results from the electron paramagnetic resonance spectroscopy, as histidine imidazole planes show a small dihedral angle in mutual plane arrangement. The observed mid-point potentials of -135 mV and -289 mV are a result of the specific geometry of heme groups and their ligands respectively. During the structure refinement process unusual high refinement R-factors were recognized, which were indicating a problem with DHC2 crystals. For this reason, an analysis of diffraction data was carried out, showing the presence of pseudo-merohedral twinning in crystals of DHC2. Two twin domains build up the crystal in a ratio of 2:1 using the twin law l, -k, h. The twinning by pseudo-merohedry is allowed due to pseudo-orthorhombic non-crystallographic symmetry. The order disorder (OD) theory describes the crystal as being composed of layers, forming the crystal lattice by translation. The weak binding energies connecting these layers are reflecting weak protein interactions, and are therefore responsible for the ambiguous translation operation, forming the crystal. These weak contacts seem to be responsible for twinning in the case of DHC2.To eliminate twinning a site directed mutagenesis study was carried out. A structural characterized mutant is showing improved diffraction behaviour, higher crystallographic symmetry and no presence of twinning. The structural model is differing from the wild-type model in the region, where the mutagenesis was carried out. The only drawback from this mutation is non-crystallographic translation symmetry having a negative effect on reflection intensities.