NMR characterization and solution structure determination of the oxidized cytochrome c ₇ from Desulfuromonas acetoxidans

Abstract

The solution structure of the three-heme electron transfer protein cytochrome c 7 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 c 3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c 7 is similar to that of three out of four hemes of cytochromes c 3 . The reduction potentials of the individual hemes, which have been obtained through the sequencespecific 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.

800 MHz 1H NMR solution structure refinement of oxidized cytochrome c7 from Desulfuromonas acetoxidans

“…With respect to the previous structure [24] the overall fold is confirmed. However, the loop 55Ϫ63 has a significantly different orientation with respect to the previously published structure and it is now better defined thanks to the increase in longrange NOEs, whereas in the previous structure was disordered and free to move due to the absence of NOEs, as it was reported [24].…”

“…An extensive sequence-specific assignment of the protein resonances in the 1 H-NMR spectrum of the fully oxidized form of cyt c 7 from D. acetoxidans was available [24]. We have repeated the assignment of the protein residues under the same experimental conditions (see above) but recording the two-dimensional NOESY maps with a higher frequency spectrometer (800 MHz), in order to obtain more structural constraints and thus a better structure definition.…”

“…Cyt c 7 from D. acetoxidans was obtained and purified as reported [24]. The 1 H-NMR samples were prepared by dissolving the lyophilized protein in 100 mM sodium phosphate, pH 6.5, to give 2Ϫ3 mM solutions.…”

“…From 600-MHz 1 H-NOESY, COSY, and TOCSY experiments a structure was obtained although only two ring protons of two of the six axial histidine rings could be assigned, being as broad as 500Ϫ 1000 Hz [24]. No signal of the other four histidine rings was assigned nor has been assigned now.…”

“…The structure of the protein backbone is such that not many long-range NOEs could be expected and short-range NOE constraints are not best suited to solve this type of structure. Despite these difficulties a family of 20 conformers was obtained with the program DIANA [26] with small violations and a backbone rmsd of 0.70Ϯ0.22 Å with respect to the average structure [24]. The extensive use of the REDAC strategy [26] was responsible for such low rmsd values.…”