Isolation and crystallization of high molecular weight cytochrome from Desulfovibrio valgaris Hildenborough

Abstract

High molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough has been isolated, purified and crystallized. The molecular weight was estimated to be 75 000 as the mean value from the results of gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains 16 c-type heme groups per molecule. The purified high molecular weight cytochrome c shows typical c-type cytochrome absorption spectra, with an α-peak at 553.2 nm ( ϵ 553.2 mM = 428) in the ferro form. The amino acid composition shows that the protein contains a sufficient number of cysteine and histidine residues to account for the high content of heme groups. The amino acid composition of D. vulgaris Hildenborough high molecular weight cytochrome c has no similarities to the D. desulfuricans hexahemoprotein nitrite reductase. The high content of the heme groups might suggest that the cytochrome has an alternate intrinsic biological function. Crystals of the high molecular weight cytochrome c have been grown from solutions of poly(ethylene glycol) 1000 or 2-methyl-2,4-pentanediol. The crystals are in space group P6 2 or P6 4 with unit cell dimensions a = b = 227.8, c = 105.7 Å and γ = 120°. A large unit cell volume of 4.75 · 10 6 Å 3 suggests that there are four or five protein molecules per asymmetric unit. They diffract to better than 4.0 Å resolution and appear to be resistant to radiation damage.