Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

Abstract

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.

Figure 1.

Sequence of ORF03300 from G. sulfurreducens; its four domains are aligned with each other.

Figure 2.

Amino acid sequence alignment of domain C of the four domain poly-c₇-type cytochrome (ORF03300) and cytochrome c₇ (PpcA) from G. sulfurreducens. Identical residues are shown in bold; the heme binding residues are shown in gray boxes and the heme numbering is indicated below. The sixth axial ligand to heme IV in both proteins is shown in a black box (note that these residues do not align in the sequence).

Figure 3.

Ribbon drawing of domain C (left) and overlap of domain C and PpcA (right). Domain C and PpcA were overlapped using heme IV atoms. Domain C, blue; PpcA, magenta; hemes in domain C, green; hemes in PpcA, gray.

Figure 4.

Stereo views showing hemes, α carbons, and side chains that bind the hemes in (top) domain C, (bottom) cytochrome c₇ (PpcA) from G. sulfurreducens. The PpcA structure shown also includes a molecule of deoxycholic acid in the lower part of the figure. Heme I is on the right, heme III is in the middle, and heme IV is located on the left side of the figures. The orientations of the hemes IV are the same in the two molecules. Note that the methionine residue that coordinates heme IV in domain C is in the left side of the figure.

Figure 5.

The cis-peptide bond observed in domain C between residues His-36 and Thr-37. Electron density in the final simulated-annealing composite omit-map generated by the program CNS is contoured at 1σ.

Figure 6.

Visible redox titrations for domain C (open squares) and PpcA (open circles) at 298 K (pH 7.9). The molar fraction of the total reduced protein was fitted to the model described by Santos et al. (1984) and Wang et al. (1991), for various solution potentials. Solid lines indicate the results of the fits for the three macroscopic reduction potentials (vs. standard hydrogen electrode), which in domain C are −185 mV, −98 mV, and -52 mV, and in PpcA are −200 mV, −155 mV, and −110 mV.