NMR characterization and solution structure determination of the oxidized cytochrome c7 from Desulfuromonas acetoxidans

Abstract

The solution structure of the three-heme electron transfer protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the protein matrix. This information is relevant for any attempt to understand the electron transfer pathway.

Figure 1

Shown is the 600 MHz ¹H NMR spectrum of Cyt c₇ from D. acetoxidans recorded in D₂O solution, 100 mM phosphate buffer (pH 6.5).

Figure 2

Number of intra-NOEs (A) and inter-NOEs (B) per residue identified in the NMR spectra. The total height of each column represents the amount of observed experimental NOEs. The open and filled bars correspond to NOE constraints that are found to be irrelevant and meaningful, respectively. In C, diagrams of the root-mean-square deviation (rmsd) values per residue with respect to the average structure calculated on the 20-structure family for backbone (▪) and all heavy atoms are reported (○).

Figure 3

Stereoview of the ribbon diagram of the solution structure of Cyt c₇ from D. acetoxidans (A) and of the x-ray crystal structure of Cyt c₃ from Desulfovibrio desulfuricans (7) (B), displayed using molscript (36). The solution structure of Cyt c₇ has been obtained from the average of the 20 solution structures of the distance geometry family, followed by energy minimization.

Figure 4

(A) Schematic representation of the sequential connectivities involving NH, Hα, and Hβ protons. The thickness of the bar indicates the NOEs intensities. In the first line, NH resonances that have been found to exchange slowly in D₂O solution are also indicated. (B) Diagonal plot of the NOEs observed between the backbone protons, which identify the secondary structure elements.

Figure 5

Stereoview of backbone and hemes of the energy-minimized average structure of D. acetoxidans Cyt c₇. The positively and negatively charged residues are shown in blue and red, respectively. The neutral residues are shown in yellow. The three-heme groups are green, and the axial histidines are yellow. The molecule orientation is the same as in Fig. 3.