Structure of the Tetraheme Cytochrome from Desulfovibrio desulfuricans ATCC 27774: X-ray Diffraction and Electron Paramagnetic Resonance Studies

Abstract

The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement [Frazão et al. (1994) Acta Crystallogr. D50, 233-236] and refined at 1.75 A to an R-factor of 17.8%. When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept [heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues]. The three-dimensional structure of D. desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes [Palma et al. (1994) Biochemistry 33, 6394-6407]. This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling. The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations. Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure.

Structural and functional characterization of cytochrome c3 from D. desulfuricans ATCC 27774 by 1H‐NMR

“…This assignment was further confirmed by the observation of a weaker NOESY cross-peak (signal b in Fig. 2C) between M121 I and M21 III (at 4.1 A) [16]. Thus, The structural assignment of the redox potentials is now complete and establishes the order of oxidation for the haems with increasing mid-point redox potential as II and I, which have similar redox potentials at pH = 8, then IV and finally III, with values approximately 100 and 160 mV higher than the first ones, respectively.…”

“…A different assignment was reported in the literature [16] based on EPR difference spectra. However, it should be noticed that this difference was performed between spectra for which the signals are overlapping and have different linewidths (Fig.…”

“…In multiredox-centre proteins, this is a crucial piece of information for understanding their mode of action since it links unequivocally specific structural features with the respective redox potentials. In the particular case of D. desulfuricans ATCC 27774 cytochrome c3, the macroscopic haem redox potentials have already been determined by EPR (-380, -370, -260, -140 at pH = 8) [16], but unfortunately the assignments deduced for the ordered microscopic haem redox potentials as well as some of the reported midpoint redox potentials are wrong, as shown in this article.…”

“…Remarkably, although the structures available for this protein from different organisms show the same spatial organisation of the four-haem core [8][9][10][11][12][13][14][15][16], the amino acid sequence homology can be as low as 23% [17]. In contrast, 64% of the conserved residues are directly involved in the binding of the *Corresponding author.…”

Cytochrome c3 from Desulfovibrio gigas: Crystal structure at 1.8 Å resolution and evidence for a specific calcium‐binding site

“…A more extensive comparison of the structural waters in the five crystal structures can now be conducted between the cytochromes c3 from Dg, DvH, DvMF, DmbN, and Dd (see Table 6 above and Table 7 in Morais et al. 1995).…”