GHP, a new c-type green heme protein from Halochromatium salexigens and other proteobacteria Gonzalez Van Driessche 1 , Bart Devreese 1 , John C. Fitch 2 , Terrance E. Meyer 2 , Michael A. Cusanovich 2 and Jozef J. Van Beeumen 1 1 Department of Biochemistry, Microbiology and Physiology, Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium 2 Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ, USA Purple phototrophic bacteria produce a large variety of soluble electron-transfer proteins, the most wide- spread and abundant of which are cytochromes c 2 , c 4 , c¢, reaction center tetra-heme cytochrome c, flavocyto- chrome c–sulfide dehydrogenase, bacterial ferredoxin, and high-potential iron-sulfur protein [1,2]. Minor components are often difficult to purify and generally have not been characterized to any extent. Some are truly soluble, but others exhibit low solubility, or they are proteolytic fragments of more abundant mem- brane-bound proteins. Cytochrome c 4 , for example, is normally membrane-bound, but is shown to be present at low concentrations in the soluble fraction of many species of bacteria. Others, such as Sphaeroides heme protein (SHP) and di-heme cytochrome c, are rarely observed, although they are also widespread. In Rho- dobacter sphaeroides, the SHP gene is associated with those for a membrane-spanning cytochrome b and the Keywords cysteic acid; cytochrome c; green heme protein; Halochromatium salexigens; mass spectrometry Correspondence J. Van Beeumen, Universiteit Gent, Vakgroep Biochemie, Fysiologie en Microbiologie, Laboratorium voor Eiwitbiochemie en Eiwitengineering, Ledeganckstraat 35, B-9000 Gent, Belgium Tel: +32 9264 5109 Fax: +32 9264 5338 E-mail: jozef.vanbeeumen@ugent.be Database The protein sequence data reported in this paper will appear in the UniProt Knowledge- base under the accession number P84848 (Received 21 February 2006, revised 14 April 2006, accepted 27 April 2006) doi:10.1111/j.1742-4658.2006.05296.x We have isolated a minor soluble green-colored heme protein (GHP) from the purple sulfur bacterium, Halochromatium salexigens, which contains a c-type heme. A similar protein has also been observed in the purple bacteria Allochromatium vinosum and Rhodopseudomonas cryptolactis. This protein has wavelength maxima at 355, 420, and 540 nm and remains unchanged upon addition of sodium dithionite or potassium ferricyanide, indicating either an unusually low or high redox potential, respectively. The amino-acid sequence indicates one heme per peptide chain of 72 resi- dues and reveals weak similarity to the class I cytochromes. The usual sixth heme ligand methionine in these proteins appears to be replaced by a cys- teine in GHP. Only one known cytochrome has a cysteine sixth ligand, SoxA (cytochrome c-551) from thiosulfate-oxidizing bacteria, which is low- spin and has a high redox potential because of an un-ionized ligand. The native size of GHP is 34 kDa, its subunit size is 11 kDa, and the net charge is )12, accounting for its very acidic nature. A database search of complete genome sequences reveals six homologs, all hypothetical proteins, from Oceanospirillum sp., Magnetococcus sp., Thiobacillus denitrificans, Dechloro- monas aromatica, Thiomicrospira crunogena and Methylobium petroleophi- lum, with sequence identities of 35–64%. The genetic context is different for each species, although the gene for GHP is transcriptionally linked to several other genes in three out of the six species. These genes, coding for an RNAse, a protease ⁄ chaperone, a GTPase, and pterin-4a-carbinolamine dehydratase, appear to be functionally related to stress response and are linked in at least 10 species. Abbreviations CID, collision-induced dissociation; GHP, green heme protein; PCD, pterin-4a-carbinolamine dehydratase. FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS 2801 di-heme cytochrome c, and is probably regulated dif- ferently from the major cytochromes [3]. One of the best characterized species in this context is the purple bacterium Allochromatium vinosum, from which six minor cytochromes have been reported, for example cytochrome c-553(550), which is a c 4 , and cyto- chrome c-551, which is a c 8 [4,5]. In this organism, we have observed a green heme protein (GHP) which we have now succeeded in purifying from the related pur- ple bacterium Halochromatium salexigens, where it is somewhat more abundant, and we have characterized it sufficiently to identify it in other genomes, either from its gene sequence or from its spectral characteris- tics. H. salexigens is interesting because it has a 10% salt optimum and has a higher sulfide tolerance than other purple sulfur bacteria studied [6]. As expected, the same major electron-transfer proteins were isolated from the soluble fraction of H. salexigens, including cytochromes c 4 , c¢, reaction center tetra-heme cyto- chrome c, flavocytochrome c–sulfide dehydrogenase, bacterial ferredoxin, and high-potential iron-sulfur protein [7], but there are some interesting proteins not previously characterized from A. vinosum or other members of the Chromatiaceae, such as the photo- active yellow protein [8]. We here present the complete amino-acid sequence and some specific characteristics of the unusual bacter- ial GHP, and show that it may be a member of a so far unknown group of class I c-type cytochromes. Results Isolation of GHP The soluble proteins from the H. salexigens superna- tant were purified as described in the Experimental procedures section. The ratio A 280 ⁄ A 420 was 0.38 for the best green-colored protein fractions. The pyridine hemochrome spectrum displayed a peak at 550 nm, indicating that GHP contains a c-type heme with an absorption coefficient of 68 mm )1 Æcm )1 Æheme )1 at 420 nm. The absorption spectrum has wavelength maxima at 420, 355, and 540 nm, as shown in Fig. 1. GHP was unaffected by the reducing agent sodium dithionite or by the oxidant potassium ferricyanide, indicating that it has either a very low or high redox potential, respectively. The usual wavelength maximum for the Soret peak of oxidized low-spin c-type cyto- chromes is 410–415 nm and that of high-spin heme is near 400 nm. In the visible region, the corresponding maxima are at 530 and 500 nm, which is unlike GHP in either case. The Soret peak of reduced low-spin cytochromes is near 416–419 nm, and there are two peaks in the visible region near 525 and 550 nm, which is also unlike GHP. Reduced high-spin proteins have Soret peaks near 420–430 nm and a single broad peak in the visible region in the vicinity of 540–550 nm, characteristics that are most similar to those of GHP. However, we were left with insufficient quantities to characterize the spectral properties further, which must await cloning and overproduction. Additional data on heme ligation will be presented below. The native size of GHP was estimated by gel filtration to be 34 kDa. SDS ⁄ PAGE, however, indicates a mass of 11 kDa. Amino-acid sequence of GHP and molecular mass The complete amino-acid sequence (Fig. 2) of GHP was determined by a combination of automated Edman degradation and tandem MS of peptides gener- ated by different digestions on apoprotein and native protein (Table 1). GHP contains 72 amino-acid resi- dues, and the measured mass (Fig. 3A) of component A (8970.3 Da) is in good agreement with the theoret- ical mass of the complete sequence containing one heme group (8968.3 Da). Component B (8840.9 Da) lacks the N-terminal glutamic acid (8839.2 Da), as pointed out in Table 1. Isoform I, represented by pep- tides S5 and S6, has the sequence ALHGQVRM. Although expressed in low amount, the presence of a second isoform (II) was found by MS analysis of pep- tide S3, originating from the nonspecific cleavage of Glu-C endoproteinase at the aspartic acid residues 34 and 42, of which the N-terminal sequence was deter- mined to be: ALHGQVYD (Table 1). There are three cysteines in the GHP. Non-modified and reduced ⁄ aminopropylated peptides generated from 250 600 Absorbance Wavelength (nm) 420 540 355 280 0.0 1.0 Fig. 1. UV-visible spectrum of green colored heme protein (GHP) in 20 m M Tris ⁄ HCl, pH 7.5. The spectrum was unaffected by the addi- tion of sodium dithionite or potassium ferricyanide. The protein con- centration was  11 l M. GHP, a new cytochrome c from H. salexigens G. Van Driessche et al. 2802 FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS the Glu-C endoproteinase digestion on the native pro- tein were deposited on a MALDI-MS target plate to determine which peptides contain a free cysteine. Pep- tides covering the sequence regions 32–44 (1457.7 Da) and 32–48 (1941.9 Da) were shown to contain a modi- fied cysteine, by a mass shift of 57 Da to 1514.7 Da and 1998.9 Da, respectively (data not shown). As expected from the conventional CXXCH heme-binding motif, the two other cysteines are involved in covalent heme binding [9]. Heme iron oxidation state As mentioned above, the UV spectrum (Fig. 1) of the GHP shows a 5–10 nm red-shift of the Soret peak, suggesting unusual heme ligation if the protein is oxid- ized low-spin. We therefore aimed to examine the nat- ure of this ligation by MS techniques. The analyses showed that heme-containing peptides were present in the chromatographic fractions S7 and S8 (Table 1) obtained after digestion of the native protein with Glu-C endoproteinase. The heme is thus covalently bound to peptides covering the N-terminal regions 1–21 and 1–31, the former originating by incomplete cleavage of a Glu-Val bond (Fig. 2). No evidence for cross-linking of the heme with other peptides of the protein has been found. The heme-containing peptides were also subjected to MALDI MS fragmentation ana- lysis, and one example of such an MS ⁄ MS spectrum is presented in Fig. 3B. For comparison, Fig. 3C shows the same type of spectrum of the heme-containing pep- tides from horse cytochrome c, Paracoccus sp. cyto- chrome c 4 and Rhodoferax fermentans cytochrome c¢, the spectra of which are identical. In general, the pro- toporphyrin group bears two negative charges, result- ing from two ionized pyrrole rings. As the central iron atom has either two or three positive charges, the heme group as a whole is either neutral or singly posi- tively charged if the heme propionates are excluded from consideration [10,11]. On the basis of the chem- ical formula C 34 H 32 N 4 O 4 Fe, a mass of 616.2 Da should be measured during mass analysis, and that is exactly what we observe in Fig. 3C. GHP, however, releases a singly charged heme ion of 617.2 Da (Fig. 3B), which suggests that the heme as a whole is neutral, because of Fe(II) and considering a proton to be responsible as a charge carrier. From these data, it appears that the heme iron from the GHP is in the Fe(II) oxidation state. Evidence for oxidized cysteine in the native protein In the MALDI mass spectrum of the non-modified Glu-C peptide 32–48, peaks appeared at differences of 16, 32, 48 and 64 mass units from the major peak at 1943.0 Da (Fig. 4A). The peak at m ⁄ z 1959.0 Da can theoretically be interpreted as the oxi- dation product of either a methionine or a cysteine to Met-SO or Cys-SOH, respectively, and the peak Fig. 2. Sequence alignment of H. salexigens GHP (HS) with homologs (http://www.jgi.doe.gov/ and http://research.venterinstitute.org/) from Oceanospirillum sp. MED92 (OS), Thiomicrospira crunogena (TC), Magnetococcus sp. Mc-1 (MC), Dechloromonas aromatica (DA), Thiobacil- lus denitrificans (TD) and Methylobium petroleophilum (MP). Identical residues are in bold. Percentage identities are presented at the right of the alignment. G. Van Driessche et al. GHP, a new cytochrome c from H. salexigens FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS 2803 at 1975.0 Da may represent Cys-SO 2 H or MetSO 2 . Proof that the mass of 1959.0 Da refers to the pep- tide containing Cys-SOH is found from the spectrum in Fig. 4B, which shows the MALDI MS ⁄ MS data for the precursor ion at 1975.0 Da. There is clear evidence for the presence of the further oxidation Table 1. Mass and sequence analysis of selected peptides obtained after digestion of purified apoprotein with trypsin (T) and Asp-N endo- proteinase (D), and of native protein with Glu-C endoproteinase (S). Amino acids identified by Edman degradation and MS tandem fragmen- tation are presented in normal and bold letters, respectively. Selected peptides are numbered following their elution during chromatography. -, Amino acid not identified during sequence analysis but already determined from other peptides; NS, no signal during MS analysis. Peptide Sequence Measured mass Calculated mass Sequence position T1 RVESLDALHGQVR 1478.8 a 1478.8 29–41 T2 AIDAQALVDQNC 2850.8 b,c 2850.3 3–28 T3 EAIDAQALVDQN 2979.9 b,c 2979.3 2–28 T4 3109.9 b,c 3110.3 1–28 T5 EYYNFEP 960.4 a 960.4 66–72 T6 M-EQNLELT-FDDQVDAVTTLLNR NS (2855.2) 42–65 D1 DQNCT- -HGSEYTR 1668.7 a 1668.7 11–25 D2 DER- - -SL 1002.5 a 1002.5 26–33 D3 DALHGQVRM 1025.5 a 1025.5 34–42 D4 DREYYNFEP 1231.8 a 1231.5 64–72 d D5 DALHGQVRMCE 1612.8 a 1612.7 34–47 D6 TTLLNREYYN- - - 1944.1 a 1943.9 57–72 D7 DDQVDAVTTLLNREYYN 2402.3 a 2401.1 53–72 D8 A GQVRMCEQN 2289.2 a 2289.1 34–52 B GQVRMCE 2746.5 a 2746.3 34–56 S1 QNLE NS (502.2) 45–48 S2 VYTRDERRVE 1321.9 a 1321.7 22–31 S3 ALHGQVYD 901.6 a 901.4 35–42 e S4 YYNFEP 831.4 b 831.3 67–72 S5 SLDALHGQVRM(Cys-SO 3 )EQNLE 1990.0 a 1989.8 32–48 S6 SLDALHGQVRMCEQNLE 1942.0 a 1941.9 32–48 S7 A AID-QALVDQ- - -G 4110.1 b 4110.1 1–31 B EAIDAQALVDQN-TG-HGSEVYT-D 3981.1 b 3981.0 2–31 C 3852.1 b 3852.0 3–31 S8 A AID-Q-LV-QN-TG 2805.8 b 2805.8 1–21 B EAIDAQ-LVDQN-TG-HGS 2676.8 b 2676.7 2–21 C 2547.8 b 2547.7 3–21 S9 LTWFDDQVDAVTTLLNRE 2134.8 a 2135.0 49–66 a MALDI. b ESI. c For complete removal of the covalently bound mercury molecule, the peptide was reduced with dithiothreitol. d Nonspecific cleavage originated by deamidation of asparagine to aspartic acid. e Peptide from GHP isoform II. 001 0 % 0 578 0029 mass (Da) mass (Da) 9.0488 3 .0798 * * * * )27-2(B )27-1(A 001 0 % 006 03 6 2.81 6 2.716 2.616 001 0 % 006 036 2.716 2.816 2.916 * * * mass (Da) A B C Fig. 3. (A) MALDI mass spectrum of native GHP. The asterisks represent an additional mass of 16 Da compared with the mass of the peaks preceding it. (B) MS ⁄ MS fragmentation of the heme-containing peptide of the GHP showing a detailed spectrum of the heme. (C) The same fragmentation analysis as in (B), but now for horse cytochrome c, as well as for Paracoccus diheme cytochrome c and Rhodoferax fermen- tans cytochrome c¢. GHP, a new cytochrome c from H. salexigens G. Van Driessche et al. 2804 FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS product Cys-SO 2 H(b 12 -b 11 ) and no evidence for the oxidation of Met-SO to methionine sulfone (Met- SO 2 ). Moreover, the small peak at 1277.7 Da refers to the species b 12 -H 2 SO 2 , due to the phenomenon of b-elimination under collision-induced dissociation (CID) conditions [12,13]. Concerning the mass peak at 1991.0 Da (Fig. 4A), the MALDI MS ⁄ MS spec- trum of the precursor ion at 1991.0 Da (Fig. 4C) shows a distance of 151 Da between the b 12 –b 11 ions, which fits perfectly the residual mass of cysteic acid (Cys-SO 3 H), an interpretation that is further supported by the peak at 1277.7 Da for the species b 12 –H 2 SO 3 . When analyzed by fragmentation analysis after electrospray ionization (ESI MS ⁄ MS) (Fig. 4D), the same precursor ion does not show a clear b 12 species, but the mass difference between b 13 –b 11 ions (280.2 Da) fits exactly the sequence cysteic acid– glutamic acid. This assignment is further supported by the loss of 82 Da for b 13 –H 2 SO 3 (at 1407.1 Da) and of 82 Da from the undetected b 12 species (expec- ted at 1360.1 Da), giving rise to b 12 –H 2 SO 3 at 1278.0 Da. Finally, the peak at 2007.0 Da in Fig. 4A can be explained by the presence of one cysteic acid and one methionine sulfoxide in the same peptide. Although there is evidence for the presence of singly oxidized methionine in the protein, it occurs only after complete oxidation of the cysteine, which is remarkable because methionines are known to be the most susceptible residues to oxidation by almost all forms of reactive oxygen species [14]. This indicates that the modification of cysteine to cysteic acid at position 43 may have occurred in vivo and that it did not originate by nonspecific oxidation during isolation or storage of the GHP. * - H SO (82 Da) from b12 and/or b13 23 100 % 0 1943.0 1959.0 1975.0 1991.0 2007.0 1937 2011 Cys-SOH Cys-SO H 3 Cys-SO H 2 Met-SO and Cys-SO H 3 100 % 0 1056 1532 1208.7 1077.6 1251.7 1472.7 1343.7 Met Glu Mass (Da) Mass A B C D (Da) 1277.7 Cys-SO H 2 b10 b11 b12 b13 -HSO 22 100 % 0 800 1800 822.5 1208.6 1077.6 1251.7 1488.7 Mass (Da) 1277.7 921.5 1616.8 1730.8 1359.6 Val Asn Arg Glu Met Gln b8 b9 b11 b13 b14 b10 b12 b15 Cys- SO H 3 1489.1 1407.1 1077.9 921.7 1844.3 1731.2 1617.2 1278.0 1208.9 1252.0 822.6 b8 b9 b10 b11 b13 b15 b14 b16 Val Asn Arg LeuMet Gln 100 % 0 800 1800 * * Mass (Da) * Cys-SO H +Glu 3 Fig. 4. (A) Detailed MALDI mass spectrum of a Glu-C peptide from sequence region 32–48 (theoretical mass 1941.9 Da); the interpretation of the molecular species follows from the spectra B–D. (B) MALDI MS ⁄ MS characterization of the precursor ion of 1975.0 Da. (C) MALDI MS ⁄ MS characterization of a precursor ion of 1991.0 Da. (D) ESI-MS ⁄ MS of the ion at m ⁄ z 997.5 Da (z ¼ 2). Ion fragments are indicated using the international convention for the nomenclature of MS fragmentation. All values represent the singly protonated species. G. Van Driessche et al. GHP, a new cytochrome c from H. salexigens FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS 2805 Discussion Sequence identification Searches using the blast engine returned six homologs, annotated as hypothetical proteins, with identities ran- ging from 38% to 44% with H. salexigens GHP, as shown in the alignment of Fig. 2. The Magnetococcus sp., Dechloromonas aromatica, Thiomicrospira crunogena and Thiobacillus denitrificans GHP homologs are more closely related to one another, at 46–64% identity. The Methylobium petroleophilum and Oceanospirillum GHP homologs, at 31–46% identity, are the most divergent on a percentage basis. The sizes of translated genes and the location of the signal peptides for the homologs indicate that the H. salexigens GHP sequence represents the whole protein and that it is not a proteolytic fragment of a larger membrane-bound protein. The sequence alignment furthermore suggests that GHP may be a class I cytochrome, as there is a consensus GxxxHxxxCxxCH heme-binding motif near the N-ter- minus and a YY motif near the C-terminus. Secondary- structure prediction (using predator) indicates that GHP from all seven species may be composed of three helices, an N-terminal helix that binds the heme (resi- dues 4–18), a central helix (residues 28–43), and a C-ter- minal helix (residues 54–67), which is consistent with the identification of GHP as a class I heme protein [1]. The usual distance of the YY motif from the methionine resi- due as the heme sixth ligand in class I cytochromes is 17 residues, although there are exceptions. This distance refers to L49 of GHP, a position that is not conserved in the GHP homologs. There is a methionine residue in H. salexigens GHP at position 42, but it is not con- served in the homologs either. Instead, they contain a conserved cysteine at position 43, which suggests that this residue may have functional relevance, either as a heme ligand or because of its presumed location near the face of the heme. Oxidation of a cysteine residue to cysteine sulfonic acid and ⁄ or sulfinic acid in nonheme iron metalloen- zymes has previously been observed in a serine ⁄ threon- ine and a tyrosine phosphatase [15,16], a nitrile hydratase [17], a cysteine dioxygenase [18] and an inacti- vated peptide deformylase [19]. The last of these is very labile under oxygen stress, resulting in the oxidation of the catalytic Fe 2+ center of the deformylase into the inactive Fe 3+ ion, accompanied by the conversion of Cys90, a ligand of the catalytic iron center, into a cys- teine sulfonic acid. On the other hand, the light-sensitive nitrile hydratase from Rhodococcus sp. N-771 is a bac- terial metalloenzyme, catalyzing the hydration of nitriles to the corresponding amides, of which Cys112 is post-translationally modified to a cysteine sulfinic acid in the native form [17]. The biological role of Cys-SO 2 H in the photoresponse and ⁄ or catalysis remains unclear. In addition to the five enzymes in which oxidized cys- teine residues appear to play a role in the catalytic activ- ity, the peroxiredoxins should also be mentioned with respect to the present discussion on GHP. Peroxiredox- ins are a family of peroxidases that reduce hydrogen peroxide and alkyl hydroperoxides to water and corres- ponding alcohols, respectively, with the use of reducing equivalents provided by thiol-containing proteins such as thioredoxin or glutathione [20]. The active-site cys- teine is selectively oxidized to cysteine sulfenic acid dur- ing catalysis. The sulfenic group is usually unstable and can be further oxidized to sulfinic acid and ⁄ or cysteic acid, leading to inactivation of peroxidase [21,22]. The presence of sulfinic acid in peroxiredoxin was originally thought to be irreversible, but is now known to be repaired by reaction with one of the two cysteines of the other subunit of the homodimer, forming a disulfide, which is subsequently reduced by thioredoxin ⁄ thi- oredoxin reductase [23,24]. Whether the presence of a cysteic acid in GHP originates by post-translational modification and ⁄ or is the consequence of the need of a higher oxidation state in the catalytic function of the protein, as it is in peroxiredoxin, will remain unanswered until evidence is found that points to its bio- logical role. Origin of the H. salexigens GHP green color and nature of heme ligation As the GHP from H. salexigens has a conventional c-type heme (616.2 Da), as determined by tandem MS analysis, and is covalently linked to the protein through the classical CXXCH sequence, we conclude that the color of the GHP is not a consequence of any covalent heme modification, but is rather the result of an unusual heme ligation or heme environment. Low-spin heme proteins are usually colored red, but high-spin proteins are either olive green or brown. We presented evidence from homology to class I cyto- chromes that the heme ligand in GHP may be a cys- teine, and if so, it should be low-spin and red in color. The absorption spectrum is most similar to reduced high-spin proteins, and MS data indicate that the heme is present in the reduced state. However, neither result is conclusive as very few proteins with cysteine ligands have been spectrally characterized and the heme could have become reduced during the isolation of the pep- tides. Furthermore, the heme should have a low redox potential if the cysteine is ionized, as in the M80C mutant of cytochrome c [25], or a high-potential if GHP, a new cytochrome c from H. salexigens G. Van Driessche et al. 2806 FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS un-ionized, as in SoxA [26]. In fact, SoxA contains two His ⁄ Cys-ligated low-spin hemes, one of which can- not be reduced by dithionite and is presumably the catalytic heme with Cys ionized, whereas the other becomes protonated upon reduction. Furthermore, a post-translational modification of the catalytic heme ligand converts it into a persulfide, which is presuma- bly related to its enzymatic role in sulfur oxidation. Whether such a modification occurs in GHP remains to be determined. It is remarkable that the M80C mutant of cytochrome c has an unusually low redox potential, a red-shifted Soret peak with lower absorp- tion coefficient than the wild-type, and a prominent delta peak, at 355 nm, similar to GHP [25]. At present, the heme environment in GHP and its sixth ligand remain speculative, although we favor a low-spin heme with an ionized cysteine ligand. Genetic organization At present, we do not have experimental evidence for the biological function of the GHP. Its identification as a heme protein, however, is important in that it elevates the homologs from being hypothetical to genes with a clear structural feature, thus improving annota- tions in future genome sequences. Furthermore, the arrangement of a gene in an operon may provide clues to the functional role of the protein under study. In Fig. 5, we can see that the context of each of the homologous GHP genes is different, although related. The Dechloromonas GHP gene overlaps with that for a GTPase (which contains a nucleotide-binding zinc fin- ger domain) by four bases on the downstream side, and overlaps a gene for a membrane-bound prote- ase ⁄ chaperone by four bases on the upstream side as well. Furthermore, the protease is in the opposite ori- entation to that of an mRNA-degrading RNAse. For most species that have these three associated genes, such as Nitrosomonas europaea and Chromobacterium violaceum, a pterin-4a-carbinolamine dehydratase (PCD) gene coding for the enzyme PCD, which regen- erates the phenylalanine hydroxylase cofactor and also serves a regulatory role, takes the place of GHP. Thio- bacillus is an exception in that the GHP gene is 11 muecaloivmuiret ca bomo rh C snacifir t inedsullicaboihT e sA NR enorepahc/esaetorP DCP esaPTG nieto r PemeHneerG e sa lor dyH esaremosiediflusid-loihT mu lihpoe lort ep mu i bo l yht e M aeaporuesanomosortiN 67EN57 EN 77EN 87EN 4- 4- 563 2VC6632V C7632V C8632 VC 851 11 4- 4- 821 27 511 9381dbT 0 48 1dbT 1481dbT 2481dbT aci tam or asano mo rolhce D 75 4- 4- 10 03o raD 2003oraD 3003oraD 4003oraD 31- 8- 5242AepM 6242AepM 7242AepM 4- 4 - 1192AepM 4192AepM2192AepM 55 3192AepM 69 424 2AepM rotalugerlanoitpircsnarT Fig. 5. Genetic organization of the GHP homologs from N. europeae, C. violaceum, T. denitrificans, D. aromatica and M. petroleophilum. The numbers above the central lines indicate the separation of genes; a minus sign refers to an overlap with an adjacent gene, and a number without a sign refers to the stretch of bases between adjacent genes. Numbers below indicate the position on the chromosomes, following the annotations from the Joint Genome Institute (http://www.jgi.doe.gov/index.html). G. Van Driessche et al. GHP, a new cytochrome c from H. salexigens FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS 2807 bases upstream of the PCD, which in turn is 158 bases upstream of the homologous GTPase gene. However, the Thiobacillus genome appears to lack the prote- ase ⁄ chaperone. The Methylobium GHP gene overlaps a thiol disulfide isomerase gene, which overlaps a hydrol- ase gene by eight bases. At another locality on the gen- ome, the homologous protease ⁄ chaperone, the PCD and the GTPase form an operon opposite the RNAse, similar to the arrangement in Dechloromonas but with the PCD replacing the GHP. Thiomicrospira crunogena GHP is not part of an operon, but is somewhat loosely associated with a protease ⁄ chaperone and DNA heli- case. The above four enzymes are clustered on another part of the chromosome (data not shown). N. europeae, Polaromonas sp., C. violaceum, and Rhodoferax ferrireducans contain the same operon structure as Methylobium, but they lack a correspond- ing gene for GHP. The PCD gene is located elsewhere on the chromosome in Ralstonia eutropha, Azoarcus sp., Methylococcus capsulatus, Bordetella pertussis, Methylobacillus flagellatus, and Burkholderia cepacia, but the RNAse, protease, and GTPase remain clus- tered. There are many other species that have these genes, although there are cases in which only two of them are organized in a cluster, such as the RNAse and GTPase in Pseudomonas aeruginosa or the RNAse and the protease in Escherichia coli. There are thus sufficient examples of these gene arrangements to indicate that their occurrence is not random. Furthermore, the GHP does not appear to have been randomly inserted in the cluster, as it displa- ces the PCD in Dechloromonas and the protease in Thiobacillus, indicating that these insertions were inde- pendent evolutionary events. The RNAse, called ‘orn’ in E. coli, is an essential gene that carries out the final step in mRNA degradation from small oligoribonucle- otides to mononucleotides [27]. The GTPase, called YjeQ in E. coli, is also an essential gene that binds to ribosomes to regulate translation [28]. The GTPase has an OB domain at the N-terminus and a zinc-finger domain at the C-terminus which are involved in bind- ing. PCD is known to have two roles, as an enzyme and as a regulator. It normally functions as a homo- tetramer, but in its regulatory role it forms heterotetra- mers to activate partner proteins [29]. In this case, the likely binding partner is the GTPase. The prote- ase ⁄ chaperone, which is called HtpX in E. coli [30], is membrane bound and helps to refold denatured pro- teins or to degrade those that cannot be refolded. It is a heat shock protein that is under the control of factor sigma 32 and a histidine kinase–response regulator pair, called CpxAR, which is only present in close rela- tives of the enteric bacteria [31]. The protease ⁄ chaper- one is generally activated when denatured proteins appear in the periplasm. From all these considerations, it appears that GHP may be part of a heat shock response, with respect to the particular biochemical function of H. salexigens perhaps being related to its capacity as a sulfur carrier. That is, denatured iron-sulfur proteins would release sulfide, which may react with GHP and be oxidized. PCD, RNAse and GTPase are known to be cytoplas- mic enzymes and, furthermore, the enzymatic site of the membrane-bound protease ⁄ chaperone is on the cytoplasmic side. Thus, GHP is the only protein in this group that is periplasmic. In the particular case of Methylobium, the thiol disulfide isomerase is periplas- mic, like GHP. The heat shock response includes both cytoplasmic and periplasmic components. In addition to thiol disulfide isomerases, proline cis–trans isom- erases, which are often periplasmic, are involved in protein renaturation and are generally included in the heat shock response. We aim to investigate the exact functional role of GHP as soon as more material becomes available after cloning, expression, genetic and physiological characterization, complemented by structural studies using X-ray crystallography. Experimental procedures Bacterial culture and protein isolation H. salexigens, strain SG 3201 (gift from P. Caumette, Uni- versite ´ de Pau, France), was grown on the recommended medium [6] at pH 7.4, with the addition of 100 gÆL )1 NaCl, 0.5 gÆL )1 sodium acetate, and 0.5 gÆL )1 sodium thiosulfate. Cells were grown phototrophically for 5 days in 1-liter pre- scription bottles to stationary phase, which resulted in a density of 2.5 g per liter culture. Some 400 g wet cell paste was suspended in 1.5 L 0.1 m Tris ⁄ HCl, pH 8.0, and soni- cated to fractionate the cells in 200-mL aliquots for 3 · 4 min at 4 °C. The suspension was centrifuged at 38 000 g for 20 min, and the supernatant was then centri- fuged at 245 000 g for 3 h. The cell-free extract from H. sa- lexigens was adsorbed on to DEAE-cellulose, and the column developed with a stepwise salt gradient. Electron- transfer proteins were eluted in the order: high-potential iron-sulfur protein (80 mm NaCl), cytochrome c¢ (180 mm NaCl), flavocytochrome c–sulfide dehydrogenase (250 mm NaCl), GHP (300 m m NaCl), bacterial ferredoxin (poorly resolved at 350 mm NaCl), and cytochrome c553 (500 mm NaCl). The GHP fraction was further purified by gel filtra- tion on Sephadex G75, by ammonium sulfate precipitation (50–70% saturation), and by chromatography on hydroxy- apatite, where it was eluted at 25 mm phosphate from a 0–100 mm gradient. Final purification was achieved by FPLC on a TSK column. GHP, a new cytochrome c from H. salexigens G. Van Driessche et al. 2808 FEBS Journal 273 (2006) 2801–2811 ª 2006 The Authors Journal compilation ª 2006 FEBS Heme removal and protein modification The covalently bound heme was removed by treatment of 90 lg native protein with mercuric (II) chloride in 6 m urea containing 0.1 m HCl, as described by Ambler & Wynn [32]. After overnight incubation at 37 °C, the reaction mixture was desalted by ultrafiltration through a Centricon-3 Protein Concentrator (Amicon, Danvers, MA, USA) with 100 mm ammonium bicarbonate, pH 8.0, as final buffer solution. Enzymatic digestions The first half of the concentrated apoprotein solution in 100 mm ammonium bicarbonate, pH 8.0, was used for digestion with trypsin (Boehringer, Mannheim, Germany) at an enzyme to substrate ratio (mass ⁄ mass) of 1 : 55 for 20 h at 37 °C. The second half of the concentrated apopro- tein was digested with 2 lg Asp-N endoproteinase (Boeh- ringer) in 100 mm ammonium bicarbonate, pH 8.0, and incubated overnight at 37 °C. Glu-C endoproteinase (Boeh- ringer) digestion was performed on 100 lg native protein at an enzyme to substrate ratio of 1 : 40 in 100 mm ammo- nium bicarbonate, pH 8.0, at 37 °C for 4 h. Peptide purification Peptides from the tryptic and Glu-C endoproteinase diges- tion on apoprotein and native protein, respectively, were separated on a Pep-S II C 2 ⁄ C 18 column (4.6 · 250 mm; Amersham Biosciences, Uppsala, Sweden) using a model 870 three-headed plunger pump, a model 8800 system controller, and a UV monitor set at 220 nm (all parts from Dupont, Wilmington, DE, USA). The generated peptides from the Asp-N endoproteinase digestion on the apoprotein were purified on a Brownlee PTC-C 18 column (2.1 · 25 mm; PerkinElmer, Boston, MA, USA) using a SMART chroma- tographic system (Amersham Biosciences). In all cases, the peptides were eluted with a gradient of 0.1% trifluoroacetic acid in water (solvent A) to 0.08% trifluoroacetic acid in 80% acetonitrile (solvent B), at an appropriate flow rate, as recommended by the manufacturer of the column. Sequence and molecular mass analyses Sequence analyses were carried out on a 477A pulsed liquid sequenator with on-line detection of the phenylthiohydanto- in-amino acids on a 120A separation system (Applied Bio- systems, Foster City, CA, USA). MS analyses were initially carried out on an ESI hybrid quadrupole-time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, UK), equipped with a nanoflow Z-spray ionization system. MS ⁄ MS of peptides using CID was performed with argon as the collision gas, at a collision energy of 20–40 eV, depending on the mass and charge state of the precursor ion. The MS ⁄ MS spectra were trans- formed using the MaxEnt Sequence Software supplied with the mass spectrometer. Before mass analysis, the HPLC fractions were dried and redissolved in 40 lL 50% acetonit- rile ⁄ water, containing 0.1% formic acid;  10 lL were used for the ESI and MS ⁄ MS analyses. The instrument was cal- ibrated with myoglobin and trypsinogen in the mass range 500–2500 m ⁄ z. The collected peptides were also spotted on to a MALDI sample target plate with a matrix consisting of a saturated solution of a-cyano-4-hydroxycinnamic acid (Sigma, St Louis, Missouri) in 50% acetonitrile ⁄ 0.1% trifluoroacetic acid. MS and MS ⁄ MS were performed in the positive re- flectron mode using a MALDI-TOF mass spectrometer (model 4700 Proteomic Analyzer; Applied Biosystems). Peptide mass spectra were obtained in the mass range 700– 4000 Da, with calibration in the default mode, and major or interesting peaks were selected for MS determination of their amino-acid sequences. Tandem mass spectra were acquired by accelerating the precursor ions to 8 keV, select- ing them with the timed gate set to a window of 6 Da, and performing CID at 1 keV. Gas pressure (air) in the CID cell was switched off, or at medium pressure, depending on the obtained fragmentation or sequence information. The mass of the native GHP was determined in the linear mode with horse cytochrome c as internal calibrant. Determination of free cysteines For the determination of free cysteines, about 500 pmol native protein was digested with 0.2 lg Glu-C endoprotein- ase in 25 mm ammonium bicarbonate, pH 7.8, at 37 °C for 1 h. Half of the digestion mixture was then reduced with dithiothreitol and treated with 3-bromopropylamine follow- ing the procedure of Jue & Hale [33]. After incubation at room temperature for 1 h, 1 lL of the reaction mixture was mixed with an equal volume of saturated a-cyano-4- hydroxycinnamic acid in 50% acetonitrile ⁄ 0.1% trifluoro- acetic acid and directly analyzed using MALDI MS techniques. The untreated half of the digestion mixture was analyzed by MS in a comparative way. 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GHP, a new c-type green heme protein from Halochromatium salexigens and other proteobacteria Gonzalez Van Driessche 1 , Bart Devreese 1 ,. and of native protein with Glu-C endoproteinase (S). Amino acids identified by Edman degradation and MS tandem fragmen- tation are presented in normal and