Preliminary crystallographic analysis and further characterization of a dodecaheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774

Abstract

Dodecaheme cytochrome c has been purified from Desulfovibrio (D.) desulfuricans ATCC 27774 cells grown under both nitrate and sulfate-respiring conditions. Therefore, it is likely to play a role in the electron-transfer system of both respiratory chains. Its molecular mass (37768 kDa) was determined by electrospray mass spectrometry. Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the cytochromes of the c(3) type from sulfate-reducing bacteria sequenced at present. The midpoint redox potentials of this cytochrome were estimated to be -68, -120, -248 and -310 mV. Electron paramagnetic resonance spectroscopy of the oxidized cytochrome shows several low-spin components with a g(max) spreading from 3.254 to 2.983. Two crystalline forms were obtained by vapour diffusion from a solution containing 2% PEG 6000 and 0.25-0.75 M acetate buffer pH = 5.5. Both crystals belong to monoclinic space groups: one is P2(1), with a = 61.00, b = 106.19, c = 82.05 A, beta = 103.61 degrees, and the other is C2 with a = 152.17, b = 98.45, c = 89.24 A, beta = 119.18 degrees. Density measurements of the P2(1) crystals suggest that there are two independent molecules in the asymmetric unit. Self-rotation function calculations indicate, in both crystal forms, the presence of a non-crystallographic axis perpendicular to the crystallographic twofold axis. This result and the calculated values for the volume per unit molecular weight of the C2 crystals suggest the presence of two or four molecules in the asymmetric unit.

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family

“…There is, however, no structural evidence that the molecule should be functional as a dimer. This model corresponds to a molecular mass of 36,811 Da, consistent with the experimentally observed value of 37,768 Da [6]. It is likely that the difference will be mainly due to the eight residues that could not be identified from the electron-density maps.…”

“…Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the presently sequenced cytochromes of the c 3 type from sulphate-reducing bacteria. The midpoint redox potentials of this cytochrome were estimated to be -69 mV, -120 mV, -248 mV and -310 mV and electron paramagnetic resonance (EPR) spectroscopy of the oxidised cytochrome shows several low-spin components with a g max spreading from 3.254 to 2.983 [6]. The early finding of 12 haems in this molecule is most probably due to the uncertainties inherent to the pyridine haemochrome method of determination that was used [5].…”

“…This cytochrome is a monomer with a molecular mass of 37,768 kDa, determined by electrospray mass spectrometry [6]. Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the presently sequenced cytochromes of the c 3 type from sulphate-reducing bacteria.…”

Redox-Bohr effect in the nine haem cytochrome from Desulfovibrio desulfuricans 27774

“…2. Previous reductive titrations obtained for 9Hcc from Dd 27774 [6] were performed under different experimental conditions from the present work and thus were not incorporated in the current analysis. Modelling two non-interacting acid Á/base centres instead of one is reasonable considering the size and structure of this molecule, in which it would be difficult to envisage a single acid Á/base group having sizeable interactions with all haems (see Fig.…”

“…A nine-haem cytochrome c (9Hcc), isolated from the SRB Desulfovibrio desulfuricans ATCC 27774 (Dd 27774) [5] is a 37.8 kDa monomeric protein [6] containing 292 aminoacid residues. The haems are all low-spin with bis-histidinyl axial coordination and are folded into two tetrahaem clusters similar to that in cyt c 3 , with one extra haem located between them (Fig.…”