Overexpression of Desulfovibrio vulgaris Hildenborough cytochrome c₅₅₃ in Desulfovibrio desulfuricans G200

Abstract

Plasmid pRC41, containing the cyf gene encoding cytochrome cSs3 from Desulfovibrio vulgaris Hildenborough, was transferred by conjugation from Escherichia coli to Desulfovibrio desulfuricans G200. The structural properties of the purified protein were studied by one-dimensional and twodimensional NMR. A heterogeneity in the folding of the cytochrome isolated from D. vulgaris Hildenborough and from D. desulfuricans G200 was observed for the oxidized form. Temperature, pH and salt-dependence studies indicated that the heterogeneity does not result from an intermediate in the protein unfolding process, but derives from two conformations which are not in dynamic equilibrium.Cytochrome c,,, is a small monoheme cytochrome (Mr 9000), which has a low oxido-reduction potential (20 mV [6]. The primary structure of cytochrome cSs3 seems to be evolutionarily homologous to more than a hundred sequences of bacterial cytochromes in the cytochrome c family. In this family the heme-binding site is found in a sequence motif Cys-Xaa-Xaa-Cys-His towards the N-terminus, and the sixth ligand is provided by a methionine residue present in the Cterminal part of the sequence. 'H-NMR studies of D. vulgaris Hildenborough and D. desulfuricans Norway cytochromes cSs3 have demonstrated that the coordination geometry at the heme iron differs depending on its oxido-reduction state. In the oxidized protein the heme iron has the same coordination geometry as in the eukaryotic cytochromes (R chirality), whereas in the reduced form a new coordination geometry was observed, similar to that in cytochrome css, from Pseudomonas or Rhodopseudomonas gelutinosa and cytochrome c5 from Pseudornonas mendocina (S chirality) [7]. This atyp-

Structural Model of the Fe-Hydrogenase/Cytochromec 553 Complex Combining Transverse Relaxation-optimized Spectroscopy Experiments and Soft Docking Calculations

“…NMR Samples-The cytochrome c 553 from D. vulgaris Hildenborough was expressed and purified from D. desulfuricans G200 as described previously (17). (18) where it affords a ͱ 2 sensitivity enhancement for kinetically stable amide groups in proteins.…”

New conformational properties induced by the replacement of Tyr‐64 in Desulfovibrio vulgaris Hildenborough ferricytochrome c553 using isotopic exchanges monitored by mass spectrometry

“…DvH wild-type and muAbbreviations." ESI, electrospray ionization; MALDI, matrix-assisted tants ferricytochrome e~,~:~ were obtained as previously described laser desorption/ionization; H/D, hydrogen/deuterium; DvH Desu(fb- [24,25]. vibrio vulgaris Hildenborough; LC, liquid chromatography; MS, mass spectrometry.…”

Effects of the Tyr64 Substitution on the Stability of Cytochrome c553, a low Oxidoreduction‐Potential Cytochrome from Desulfovibrio vulgaris Hildenborough

“…Paramagnetic metalloproteins have characteristic spectra due to These different forms were noticed in the N M R spectra of the oxidized cytochrome (Fig. 3) and were found associated with a duplication of the resonances of most of the polypeptide chain protons (Blanchard et al, 1993). The minor conformations were found in different amounts for the different mutants of cytochrome c, ,.…”

“…The purification procedure of native D. vulgaris cytochrome c551 from D. desuljuricans G200 was previoudy reported (Blanchard et al, 1993 350 g wet cells were obtained from 300 1 fermentation in Postgate medium C supplemented with 0.28 mM kanamycin and were harvested as previously described (LeGall et al, 1965). The mutants.…”

“…The small size of cytochrome cSs3 relates it to the cytochromes c8, but the typical proline fragment around the axial methionine is lacking. Recent structural investigations on Desulfovibrio vulgaris ferrocytochrome and ferricytochrome c553 using 2D NMR and X-Ray crystallography (Nakagawa et al, 1990) have demonstrated that this cyto- Spontaneous Nal' derivative of D. desulfuricans GlOOA (Weiner et al, 1988) @Sod lacZAM15 e n d l recA1 hsdRI7(r;m:) supE44 thi-1 2 girA96 relAI F' A(lacZ4-argF) U169 (Hanahan, 1985) contains the cyf gene of D. vulgaris Hildenborough on a 736-bp EcoRI-Hind111 insert in pJRD215 (Blanchard et al, 1993) IncQ group, broad-host-range cloning vector: Km' Sm' (Davison et al, 1987) contains the cyfgene carrying the Y64X mutation in pJRD215 where X = V, S, F or L, this work chrome which shows only 28% identity to Pseudomonas aeruginosa cytochrome cS5,, is a structurally different cytochrome. Three conserved helices (N-terminal and C-terminal helices and a central helix) are found in the heme environment with an orientation similar to that generally observed in either mitochondria1 or bacterial cytochromes.…”