March 15, 1989 Pages 670-676
l.xMPARATIvEsTDDIRsoBPOL~c ~OREfjCItiOL&T'ElbFROIYDESDLEWVIiRtIO lWA%RIS(RILDRRBOROUQi)ARDD~VIBRIODEsfTLpDRI CARS (RORWAY)
rr. mm*, F. (ztmmBQuIw*, P. BIARCO-, J.KAL&DJIAR*audM3RUSCRI*
Laboratoire de Chimie Bacterienne du C.N.R.S., B.P. 71 13277 MARSEILLE CEDEX 9 PRANCE
-x-E
Laboratoire de Chimie et Electrochimie des Complexes. Laboratoire de Chimie Bactkrienne du C.N.R.S., Universite de Provence,
Place Victor Hugo, 13331 MARSEILLE CEDEX 3 PRANCE
Received December 19, 1988
Cytochrome c (M 26,000) has been characterized in Desulfovibrio vulgaris (Hildenb&ougg) and its properties compared with polyhemic cytochromes c isolated from the same organism and from D. desulfuricans (Norway). It can be described as an octaheme cytochromec constituted of two identical subunits. Absorption spectrum is similar ta cytochrome c (Mr 13,000) and individual redox potentials have an average value of -180 mV?
The N terminal sequence is compared with an homologous cytochrome isolated from & desulfuricans Norway. 0 1989 Academic Press, Inc.
Low potential multihemic cytochromes c have been found and investigated mainly in sulfate reducing bacteria belonging to the genus Desulfovibrio. They are cytochrome c 3 (Mr 13,000) (1) an original group of c type cytochromes characteristic of the genus Desulfovibrio and a higher molecular weight cytochrome c3 (Mr 26,000) (2-4).
Cytochrome c3 (M, 13,000) is a four heme periplasmic protein which acts as a natural electron donor and acceptor for hydrogenase. Each heme group is covalently bound to two cysteine side chains but differs from other classes of heme c proteins by the iron coordination which is of bis histidinyl type.
Alignment of the'amino acid sequences of different cytochromes c 3 points out that only 28% amino acids remain constant, essentially located in the heme binding sites (5). The tertiary structures of L desulfuricans Norway (6) and - D. vulgaris Miyazaki (7) cytochromes c show that the relative arrangement of the four hemes in the two molecules3is highly conserved (8). Each heme exhibits an individual redox potential (9), has the same iron atom axial ligands, and a local environment somewhat different.
The other type of multihemic cytochromes was first described by Bruschi et al. (1969) (2) and Ambler et al. (1971) (10) from D. gigas and D. vulgaris - 0006-291X/89 $1.50
Copyright 0 1989 by Academic Press, Inc.
Vol. 159, No. 2, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hildenborough. This 26 kDa cytochrome is indistinguishable from the 13 kDa
cytochrome c3 except in size and amino acid composition (2). Because there is evidence of gene doubling in the 13 kDa cytochromes c 3 (11) it was possible to postulate that the 13 kDa cytochrome c3 gene was at one time duplicated to form the 26 kDa protein (12). Characterization of an homologous cytochrome c3 (Mr 26,000) in D. -- d. Norway has shown that removal of hemes produced two identical monomers different from cytochrome c 3 (Mr 13,000). The presence of two types of cytochromes c
3 in the bacteria has probably a physiological importance. In & gigas, cytochrome c3 (Mr 26,000) has been described as being implicated in the reduction of thiosulfate (2). Neither these two different classes of multihemic cytochromes can replace the other in their respective reactions (13). Recently, preliminary X-ray studies of the tetraheme cytochrome c
3 and the octaheme cytochrome c 3 from D. _ gigas have been published (14). Another polyhemic cytochrome described as a unique subunit of 70 kDa molecular weight has been isolated from L vulgaris Hildenborough and crystallized (15).
In this communication, we propose a comparative study of cytochromes c (Mr 13,000) and (Mr 26,000) from L k Hildenborough and k & Norway and a 3 determination of the individual redox potentials of these polyhemic cytochromes c in order to increase our understanding of structure and function relationships of the cytochrome c 3 family.
Organism and growth conditions
D. v. Hildenborough (National Collection of Industrial Bacteria no 8303) and D.-- d. Norway 4 (N.C.1.B no 8310) were grown in the medium of Starkey (16) and Ervzted as previously described (17).
Purification of D. v. Hildenborough cytochrome c (M 26,000)
All steps were performed at + 4W and all%lifers were at pH 7.6 except potassium phosphate buffer pH 7.0.
The bacterial extract was prepared with a French pressure cell and centrifuged at 35,000 rpm for 1 hour. The supernatant was stirred overnight with 150 ml of Amberlite CC50 where cytochrome c (M 13,000) was adsorbed.
Cytochrome c (M 13,000) was completely discarded a%ter an Amberlite CG50 column ( 4~ 32 cm)? The unadsorbed fraction was stirred with 100 ml of Silica gel (Merck) for four hours. Cytochrome c (M 9,000) was adsorbed on the gel and then separated. Finally cytochromJ'%& T)r M 26,000) was adsorbed on an Alumine BIB (2,s x 5 cm) column (1OmM tris- Cl then eluted with 1M potassium phosphate buffer, and loaded into a Sephadex G50 column (4,s x 100 cm) equilibrated with 1OmM tris-HCl buffer. The cytochrome solution was passed through Amberlite CC50 (3 x 7 cm) 1OmM tris-AC1 buffer and the cytochrome fraction eluted with a non linear gradient of tris-HCl (lo-2OOmM). The final step was a carboxymethyl cellulose column (2,s x 5 cm) buffer. 28 mg of pure
~?%$r~~~ffCi%ien% was Als3 - A570 / Ait
(M 26,000Jd coulr$dbe obtained from 700 g of wet cells. The
= 3.4
Cytochrome c (M 13,000) from D. v. Hildenborough and from D. d. Norway were prepared res$ectfvely according20 n7) and (18)and D. d. NorEyTytochrome c3
(Mr 26,000) as previously described (4) with a purity coefficient of 3.2.
Determination of molecular weight
The molecular weight was estimated by analytical gel electrophoresis on 13% polyacrylamide gel- in the presence- of sodium dodecyl sulfate (SDS) according to Lugtenberg et al. (19) and determined by analytical ultracentrifugation as previously described (20). These assays were performed at 25OC in a Spinco-Beckman ultracentrifuge, model E, with speed and temperature controls. The partial specific volume value used is that of cytochrome c : 0.72 ml/g.
Spectrophotometric studies
Visible and ultraviolet absorption spectra were determined with a PU 8820 spectrophotometer Philips. The molar extinction coefficients of the cytochromes were obtained by measuring the optical densities values at their absorption maxima for solutions of known protein concentration calculated from amino acid analysis.
Determination of isolectric point
The isoelectric point was determined by isoelectric focusing (21) on a LKB Multiphor apparatus.
Amino acid analysis
Amino acid analysis were performed on a LKB 4150 amino acid analyzer.
Proteins samples were hydrolyzed in 6N HCl at llO°C for 18 h, 24 h, 48 h according to the method of Moore and Stein (22).
Sequence determination
Sequence determinations were performed in an Applied Biosystems A 470 gas-phase sequencer with 1 nmole of carboxymethylated apoprotein. The quantitative determination of phenylthiohydantoin derivates was compared to known amounts of appropriate standards by KPLC (high-pressure liquid chromatography, Waters) as described by Bonicel et al. (23).
Electrochemical technique
Electrochemical measurements were made in 0.5 M Tris-HCl buffer at pH 7.6 which also served as supporting electrolyte. Other experimental details are given in a previous paper (27). All potentials are referred to the normal hydrogen electrode (NAE).
REXILTS AND DISCUSSIOI?
The term cytochrome c
3 has been proposed to include all proteins of similar electronic absorption spectra, redox potential and thus iron-ligand field, regardless of protein size or heme content. The IUB subcommittee on cytochrome nomenclature reported by Dervartanian (25) has proposed to include the presumably 26 kDa octaheme proteins in the cytochrome c
3 class with the name cytochrome c 3 (Mr 26,000). In the present work, the comparative studies of cytochrome c 3 (Mr 26,000) from several strains of Desulfovibrio show clearly that they are of cytochrome c
3 type but composed of two identical subunits, the molecular weight of the subunit being somewhat different from one strain to another.
By analytical ultracentrifugation of cytochrome c3 (Mr 26,000) from D. -- v.
Hildenborough the molecular weight calculated is estimated to be about 43,300 + 2,000 Da. The purified protein gave a major band of 70 kDa molecular weight and another band of 20 kDa molecular weight on SDS polyacrylamide gel
Vol. 159, No. 2, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLEI : Comparison between some properties of the multiheme cytochromes from
k vulgaris Hildenborougb and L desulfuricans Norway 4”
Bacteria k desulfuricans Norway k vulgaris Hildenborough
Cytochrome c3 Molecular weight
(wr 13,000) (Mr 26,000) (Mr13,000) (Mr 26,000) 15,065 (c) 32,000 (e) 13,100 (c) 43,300 (a)
Isoelectric point 7.0 4.8 10.5 9.2 (a)
(b) (a) (d)
Redox potentials El01 - 165 - 210 - 290
E'o(mV)/NRE 32 - 305 - 270 - 335
E’O3
- 365 - 325 - 345 - 180 (a)
04 - 400 - 365 - 375
spectral data
molaf extinction coefficients
(j M cm 410 1
553:, 452 105 916 504
115 100 687 018
245 200 150 980 88?& 182 258 a) present work b) electrochemical measurements from (27) c) from sequence data d) electrochemical measurements from (9) (e) from (4).
electrophoresis. After the removal of the hemes by mercuric chloride (24) a molecular weight of 18 kDa was found for the apoprotein. We could conclude that the cytochrome is a dimer of two 18 kDa subunits. An average value of 20 1cDa from the two methods was assumed to be the molecular weight of the subunit including 4 hemes. Both subunits were strongly associated and cannot be dissoci.ated by denaturing agents such as 81-f urea or 6M guanidine. This cytochrome is similar to other cytochromes c 3 (Mr 26,000) already described in D. d. Norway (4) and D. gigas (2) but with a longer polypeptide chain.
--
Comparison of molecular weight, isoelectric point and amino acid composition of cytochrome c3 (Mr 13,000) and cytochrome c3 (Mr 26,000) from the two different strains studied is presented in Tables I and II. It can be seen that both cytochromes c3 (Mr 26,000) possesses the required number of cysteine (16 residues) and histidine (16 residues) to bind eight hemes in the dimeric form.
N terminal sequences of cytochromes c 3 (Mr 26,000) f rom D. d. Norway and D. v. -- -- Hildenborough were identified and compared to cytochrome c 3 (Mr 13,000) from the same organism (Fig. I). In the 30 first residues, only 3 residues are conserved through the four sequences. However, if we compare the N-terminal region of k 2 Hildenborough cytochrome c3 (Mr 26,000) and & & Norway cytochrome c 3 (Mr 13,000), 9 residues remain unchanged. This comparison underlines the poor amino acid sequence homology between cytochromes c 3 ("r 13,000) and (Mr 26,000).
Absorption spectrum of cytochrome c3 (Mr 26,000) is similar to cytochrome c (Mr 13,000) : the 695 nm band which is a marker of histidine-methionine 3 coordination of the heme iron is absent. All these cytochromes c
3 are bis histidine coordinated. Molar extinction coefficients of these multihemic
TABLE II : Amino acid composition of cytochrome (Mr26,000) from D. vulgaris - Hildenborough an
Amino acid composition D. desulfuricans Norway D. vulgaris Hildenborough Cytochrome c3 $ 13,000) (MT 26,000) 711, 13,000) (Mr 26,000) Lysine
Histidine Arginine Tryptophane Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine
Isoleucine Leucine Tyrosine Phenylalanine
17 8 1 0 12 11
!3 8 9 13 : i 5 i
16 18 10 ND 22 24
;:
12 12 10 16 16 6 12 6 8 12
20 9 1 0 12 2 : 10 9 i 0 3 2 3 2
30 16 14 ND 30 16 14 30 20 28 36 16 16 10 12 16 4 10
Total residues 118 250 107 318
cytochromes are compared in Table I. The absorption values of eight hemes by dimeric molecule are comparable to the values found for the four hemes in cytochrome c3 (Mr 13,000).
Cyclic voltammetry of cytochrome c 3 (Mr 26,000) from L & Norway has shown that the electrochemical system is reversible. A typical DPP curve is given in Fig. 2A, with one shoulder at Ep = - 25OmV and one peak at Ep = -320 mV. Since the electrochemical system is fast the theoretical treatment described in previous work (27) may be used to determine the redox potential values (Table
1 5 10 15 20
Cyt. c3 (Up 13,000) k & Norway : Ala-Asp-Ala-Pro-Gly-Asp-Asp-Tyr-Val-Ile-Ser-Ala-Pro-Glu-Gly-~et-Lys-Ala-Lys
Cyt. c3 (Mr 26,000) k & Norway : Glu-Thr-Phe-Glu-Ile-Pro-Glu-Ser-Val-Thr-~et-Ssr-Pro
Cyt. c3 (Hr 26,000) D. vulgaris H: Lys-Ala-Leu-Pro-Glu-Gly-Pro-Gly-Glu-Lys-Arg-Ala-Asp-Leu-Ile-Glu-Ile-Gly-Ala-Het
Cyt. c3 (gp 13,000) 0. vulgaris H: Ala-Pro-Lys-Ala-Pro
25 30 35
Cyt. c3 (Mp 13.000) L & Norway : Pro-Lys-Gly-Asp-Lys-Pro-Gly-Ala-Leu-Gln-Lys-Ihr-Val-Pro-Phe-Pro-His . . . . . (5)
- - -
Cyt. c3 (Mp 26.000) k & Norway : Lys-Gln-Phe-Glu-Gly-Tyr-Thr-Pro-Lys-Lys-Gly-Asp-Val-Thr-Phe-Asn-His . . . . . (4)
- - -
Cyt. c3 (Hr 26.000) D. vulgaris H: Glu-Arg-Phe-Gly-Lys-Leu-Asp- - -Leu-Pro-Lys- - -V&-Ala-3-Arg-I& . . . . .
Cyt. c3 (Mp 13,000) D. vulgaris H: Ala-Asp-Gly-Leu-Lys-~et-Glu-Ala-Thr-Lys-Gln-Pro-~-Val-~-Asn-His . . . . . (11)
-
FIGURE 1 : Comparison of the N-terminal region of multiheme cytochromes from
Desulfovibrio vulgaris Hildenborough and D. desulfuricans Norway.
Residues common to all proteins are underlined.
Vol. 159, No. 2, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(A)
T
2onA
A -100 1 -300 -500 E(mV) %O 0 -200 -400 &W
FIGURE 2: Differential pulse polaro~ram of :
(A) 50 @! D. hesulf&ic& Norway cytochrome c (M 26,000) (B) SO pM 5. vu aris Hi~denborough cytochrom~ cSr(M, 26,000)
y-F---
in 0,s M trls-HC pH 7.6.
Drop time : 5 s.
I) which are in good agreement with those resulting from EPR measurements (26). Polarograms of L k Norway cytochrome c3 (Mr 26,000) are clearly distinct from that of the cytochrome c 3 o$. 13,000). Calculations have shown that DPP curves result from the contribution of four pairs of identical hemes, with redox potential values different from that obtained for cytochrome c
3 ("r 13,000). The separation between the highest and the lowest individual redox potential values is of 235 mV for cytochrome c,
3 (lfr 13,000) and of only 155 mV for cytochrome c3 (?$ 26,000).
DPP curve (Fig. 3) and cyclic voltammogram relative to 5 vulgaris cytochrome c3 (Mr 26,000) d enote the existence of marked adsorption phenomena at the mercury electrode. Modification of polarograms with time accompanied by the formation of precipitate has been observed. Peak (1) in Fig. 2B is an adsorption pre-peak; peak (2) can be assigned to the reduction of heme centers. It seems that the individual redox potentials must have an average value of about -180 mV, which is less negative than that obtained for D. d. -- Norway cytochrome c3 (Mr 26,000).
The primary structure and redox properties of cytochrome c 3 (Mr 26,000) unambigously show that the molecule is a dimer in which the four hemes in each subunit are equivalent.The biochemical function of this class of high molecular weight cytochrome c
3 is different from cytochrome c 3 (Mr 13,000) and can be related to the noteworthy high heme content and the strong association of the dimeric molecule. The comparison of the spatial arrangement of the heme groups and of the polypeptide chain between cytochromes c 3 ("r 13,000) and (Mr 26,000) should give information on intra and inter heme-heme interactions and should explain the different affinity of cytochromes c 3 ("r 13,000) and (hi, 26,000) for their redox partners.
ACKNOWLEDGMENTS
The authors are grateful to J. Bonicel and M. Woudstra for the determination of the N-terminal sequences. They thank also P. Sauve for the analysis by analytical ultracentrifugation, G. Leroy and A. Dolla for their skilful discussions.
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