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• W2071434293 abstract "In vitro formation of Hydrogenobacter thermophilus cytochrome c552 has previously been demonstrated (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872–7876). Now we report that the single cysteine variants of H. thermophilus c552, which bind heme via a single thioether bond, also form in vitro. Furthermore, reaction of the apocytochromes containing either AXXCH or CXXAH in the binding motif with 2-vinyldeuteroheme and 4-vinyldeuteroheme resulted predominantly in covalent attachment between Cys-11 and the 2-vinyl moiety and Cys-14 and the 4-vinyl functionality. This observation shows that the covalent attachment of heme to apocytochrome is stereoselective, indicating that the initial non-covalent complexes between apoprotein and heme have to be in the correct stereochemical orientation for preferential promotion of thioether bond formation. Additionally, the heme derivatives 2-vinyldeuteroheme and 4-vinyldeuteroheme were reacted with wild-type H. thermophilus c552 to yield another modification of cytochromes containing only one thioether bond. These results show that the formation of the two thioether bonds in typical c-type cytochromes can occur independently from one another. Aspects of rotational isomerism of heme in heme-proteins are discussed. In vitro formation of Hydrogenobacter thermophilus cytochrome c552 has previously been demonstrated (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872–7876). Now we report that the single cysteine variants of H. thermophilus c552, which bind heme via a single thioether bond, also form in vitro. Furthermore, reaction of the apocytochromes containing either AXXCH or CXXAH in the binding motif with 2-vinyldeuteroheme and 4-vinyldeuteroheme resulted predominantly in covalent attachment between Cys-11 and the 2-vinyl moiety and Cys-14 and the 4-vinyl functionality. This observation shows that the covalent attachment of heme to apocytochrome is stereoselective, indicating that the initial non-covalent complexes between apoprotein and heme have to be in the correct stereochemical orientation for preferential promotion of thioether bond formation. Additionally, the heme derivatives 2-vinyldeuteroheme and 4-vinyldeuteroheme were reacted with wild-type H. thermophilus c552 to yield another modification of cytochromes containing only one thioether bond. These results show that the formation of the two thioether bonds in typical c-type cytochromes can occur independently from one another. Aspects of rotational isomerism of heme in heme-proteins are discussed. c-type cytochromes are found in almost all organisms and are mainly involved in electron transport. They contain at least one characteristic CXXCH motif, whereby the conserved cysteine residues are involved in forming a covalent thioether bond between the thiol functionalities and the vinyl groups of the prosthetic heme group (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar, 2Pettigrew G.W. Moore G.R. Cytochromes c: Biological Aspects. Springer-Verlag, New York1987Google Scholar, 3Moore G.R. Pettigrew G.W. Cytochromes c: Evolutionary, Structural, and Physicochemical Aspects. Springer-Verlag, New York1990Google Scholar, 4Scott R.A. Mauk A.G. Cytochrome c: a Multidisciplinary Approach. University Science Books, Mill Valley, CA1995Google Scholar, 5Barker P.D. Ferguson S.J. Structure. 1999; 7: R281-R290Google Scholar). The structure of heme and its derivatives mesoheme and deuteroheme are given in Fig. 1. Cytochrome c maturation in cells is a catalyzed process and involves complex protein systems in bacteria (6Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Google Scholar), but a single heme lyase protein in mitochondria from some cells (7Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Google Scholar). In our previous work (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar), we reported the first in vitro formation of a c-type cytochrome containing two thioether bonds. Cytochromes containing only one thioether bond are rare (5Barker P.D. Ferguson S.J. Structure. 1999; 7: R281-R290Google Scholar). However, engineered variants of the CXXCH binding motif, replacing one cysteine with an alanine residue by site-directed mutagenesis, for a bacterial cytochrome c (8Tomlinson E.J. Ferguson S.J. J. Biol. Chem. 2000; 275: 32530-32534Google Scholar) and for mitochondrial yeast iso-1-cytochrome c (9Rosell F.I. Mauk A.G. Biochemistry. 2002; 41: 7811-7818Google Scholar) have aided the understanding of this class of proteins and the requirement for covalent attachment of the prosthetic group to the polypeptide. In this work we report the in vitro formation of heme-attached AXXCH and CXXAH variants of Hydrogenobacter thermophilus c552 from the corresponding apocytochrome and heme. Furthermore, we study the reaction of the apocytochromes with the heme derivatives 2-vinyldeuteroheme (2-VDH) 1The abbreviations used are: 2-VDH, 2-vinyldeuteroheme; 4-VDH, 4-vinyldeuteroheme. and 4-vinyldeuteroheme (4-VDH). The latter two molecules are thus hybrids between heme and deuteroheme (Fig. 1). Wild-type, C11A, and C14A variants of H. thermophilus c552 were expressed and purified as previously described (8Tomlinson E.J. Ferguson S.J. J. Biol. Chem. 2000; 275: 32530-32534Google Scholar, 10Tomlinson E.J. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5156-5160Google Scholar). Apocytochromes c were prepared analogously to the method of Fisher et al. (11Fisher W.R. Taniuchi H. Anfinsen C.B. J. Biol. Chem. 1973; 248: 3188-3195Google Scholar). After the removal of the Ag+ by addition of dithiothreitol, the proteins were dialyzed extensively in sodium phosphate buffer (pH 7.0, 20 mm). SDS-PAGE analysis was carried out using the buffer system described by Laemmli (12Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar); heme activity staining was achieved by the method of Goodhew et al. (13Goodhew C.F. Brown K.R. Pettigrew G.W. Biochim. Biophys. Acta. 1986; 852: 288-294Google Scholar). The concentration of apocytochrome was determined using the extinction coefficient at 280 nm of 15.2 mm–1 cm–1 (calculated using the protein sequence) for wild-type and both single cysteine mutants (C11A and C14A) of H. thermophilus c552. Heme derivatives were synthesized as described previously (14Smith K.M. Fujinari E.M. Langry K.C. Parish D.W. Tabba H.D. J. Am. Chem. Soc. 1983; 105: 6638-6646Google Scholar). For the heme nomenclature the Fischer system of nomenclature is used. H. thermophilus apocytochrome c552 wild-type, C11A, and C14A variants (5 μm) were incubated with heme and derivatives thereof (5 μm) in 50 mm sodium phosphate buffer, pH 7.0, at 25 °C. Samples were reduced by the addition of both disodium dithionite and dithiothreitol to a final concentration of 5 mm. Solutions were thoroughly sparged with humidified argon, and reactions were carried out in the dark. Apocytochrome Production and Characterization—C11A and C14A variants of H. thermophilus cytochrome c552 were shown to be pure by SDS-PAGE analysis (Fig. 2, lanes a). The apocytochromes were shown to be devoid of heme as judged by SDS-PAGE analysis using heme staining procedures (Fig. 2, lanes b), whereas the presence of protein was shown by staining the gels with Coomassie Blue after the heme staining had been carried out. The absence of characteristics of cytochrome in the visible spectra and the disappearance of the cytochrome c fold as shown by CD analysis (data not shown) were also indicative of the removal of the covalently bound heme from the protein. In Vitro Thioether Bond Formation with Heme—Mixing either C11A or C14A variants of H. thermophilus apocytochrome c552 with ferrous (Fe(II)) heme in the presence of dithionite at pH 7.0 resulted in an increase in absorption around 424 nm relative to that of heme alone; the same trend was observed around 528 and 559 nm, bands characteristic of the presence of a reduced cytochrome (Fig. 3 for the C11A variant). The pyridine hemochrome spectrum of this mixture had its α-band at 556 nm, indicating that the heme contained two unreacted vinyl groups. These results show that the apoprotein and heme initially form a b-type cytochrome, in which the heme is not covalently attached to the peptide but in which its iron atom is coordinated by two amino acid side chains from the protein. This intermediate formed quantitatively to yield a 1:1 heme-protein complex for the C11A protein. In the case of the C14A protein, a fraction of the apoprotein (up to 40%) was unable to bind heme for unknown reasons. These results are consistent with previous observations on the wild-type protein and the AXXAH mutant, which were both shown to form a b-type cytochrome initially (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar, 10Tomlinson E.J. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5156-5160Google Scholar). Following formation of the b-type cytochrome from the mixture of C11A or C14A variants of H. thermophilus cytochrome c552 with heme in reducing conditions, the maximum at 560 nm in the protein absorption spectrum progressively shifted to either 556 or 557 nm, respectively (Fig. 3 shows the spectrum for the C11A variant). After 15 h, the pyridine hemochrome spectrum of the purified reduced protein had a resolved band at 553 nm, characteristic of a reaction with one vinyl group of the heme (3Moore G.R. Pettigrew G.W. Cytochromes c: Evolutionary, Structural, and Physicochemical Aspects. Springer-Verlag, New York1990Google Scholar, 8Tomlinson E.J. Ferguson S.J. J. Biol. Chem. 2000; 275: 32530-32534Google Scholar). These observations decisively indicate the formation of a thioether bond between heme and polypeptide, an interpretation that was further substantiated by heme staining of the proteins on SDS-PAGE gels (Fig. 2, lanes d), including first treating the heme-containing protein with acidified acetone. In the latter procedure, non-covalently bound heme dissociates from protein but covalently bound heme does not. The visible absorption spectra of the ferrous forms of the in vitro-synthesized cytochromes c, together with their reduced pyridine hemochrome spectra (Table I), were almost identical to those of the holoproteins produced in the cytoplasm of Escherichia coli (Table I and Fig. 3 for the C11A variant). The data show that these two forms, in vivo and in vitro, of the cytochromes c are almost identical with respect to both the environment of the heme and the heme modification.Table IAbsorption maxima of various species derived from Hydrogenobacter thermophilus cytochrome c552Wavelength/nmProtein speciesReduced proteinPyridine hemochrome (α band)AXXCH mutantHeme b-type complex424.5528.5559.5556Mesoheme complex415520549546Holo produced in vitro421.5524.5557553Holo produced in vivoaThese currently obtained values differ slightly from those obtained in Ref. 8.421.5525557.55532-VDH b-type complex4195225545502-VDH c-type complexbNo covalent product; pyridine hemochrome reflects unmodified heme derivatives.549.54-VDH b-type complex417522552.55504-VDH c-type complex414519.5549547.5CXXAH mutantHeme b-type complex424528559556Mesoheme complex415519.5549.5546Holo produced in vitro421525.5556553Holo produced in vivoaThese currently obtained values differ slightly from those obtained in Ref. 8.420525555.55532-VDH b-type complex418.55235545502-VDH c-type complex416519.5552.55484-VDH b-type complex416.5522552.55504-VDH c-type complexbNo covalent product; pyridine hemochrome reflects unmodified heme derivatives.550Wild-type CXXCH2-VDH b-type complex418.55235545502-VDH c-type complex415519552547.54-VDH b-type complex418522552.55504-VDH c-type complex414519.5548.5547a These currently obtained values differ slightly from those obtained in Ref. 8Tomlinson E.J. Ferguson S.J. J. Biol. Chem. 2000; 275: 32530-32534Google Scholar.b No covalent product; pyridine hemochrome reflects unmodified heme derivatives. Open table in a new tab The initial rate of thioether bond formation was very similar to that observed for the reaction of heme with wild-type H. thermophilus cytochrome c552 containing a CXXCH motif (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar). However, because of the smaller difference between the visible spectra of non-covalently and covalently bound heme-protein complex, it was not possible to elucidate unambiguously whether a biphasic kinetic behavior is observed. For the wild-type protein the biphasic kinetic profile was attributed to the heme inversion relative to its α,γ mesoaxis within the heme-binding pocket of the protein. An analogue of the b-type cytochrome intermediate formed quantitatively on the addition of reduced Fe-mesoporphyrin to reduced apocytochrome variants. The product had visible absorption maxima around 549, 519, and 416 nm (Table I) and did not heme stain on an SDS-PAGE analysis (Fig. 2, lanes c for the C11A and C14A variant). Mesoporphyrin has ethyl groups in the positions of the vinyl groups of protoporphyrin and therefore cannot form thioether bonds with the polypeptide. There was no evidence for any other type of covalent attachment of mesoporphyrin to apoprotein. In Vitro Thioether Bond Formation with 2-VDH and 4-VDH—To elucidate whether the reaction of the vinyl groups of heme with the cysteine residues of the apoproteins is selective with respect to the α,γ mesoaxis of heme, thioether bond formation was studied with mono-vinyl heme derivatives in combination with single cysteine mutants of H. thermophilus cytochrome c552. When either heme derivative (2-VDH and 4-VDH) was added to the C11A mutant of H. thermophilus apocytochrome c552 in the presence of dithiothreitol and dithionite, stoichiometric formation of a b-type cytochrome could first be observed (Table I and Fig. 4, a and b for addition of 2-VDH and 4-VDH, respectively). Very similar spectra have been obtained for synthetic cytochromes obtained from addition of mono-vinyl heme derivatives to peptides known to form cytochrome b maquettes (15Kalsbeck W.A. Robertson D.E. Pandey R.K. Smith K.M. Dutton P.L. Bocian D.F. Biochemistry. 1996; 35: 3429-3438Google Scholar). Following addition of the C11A mutant from H. thermophilus cytochrome to 2-VDH, the visible spectrum obtained remained unchanged for 15 h, as did the pyridine hemochrome spectrum, which had an α-band around 550 nm, which is the average of the pyridine hemochrome maximum of the α-bands of heme (containing two vinyl groups in the 2- and 4-position) and deuteroheme (containing two hydrogens at the 2- and 4-position). Both these observations suggested that no reaction had occurred between the Cys-14 and the 2-vinyl moiety of the heme derivative. The SDS-PAGE analysis, including heme staining methodology to test for covalently bound heme, suggested that very little attachment of 2-VDH to the apoprotein had occurred (Fig. 5, lane 4). The spectrum of the mixture of the C11A mutant of H. thermophilus apocytochrome c552 and 4-VDH, however, had changed after 15 h (Table I and Fig. 4b). The pyridine hemochrome spectrum had an α-band around 547.5 nm. These experimental data suggested that a reaction between the 4-vinyl group of 4-VDH and the Cys-14 of the apocytochrome had taken place, which was confirmed by heme staining in conjunction with SDS-PAGE analysis (Fig. 5, lane 5) showing that heme was attached to the protein. Approximately 80% of b-type cytochrome intermediate reacted to form covalently attached heme-protein as judged by pyridine hemochrome analysis. It is unclear why the reaction was not stoichiometric as it was with heme itself. The reason for the slightly lower yield of covalent complex between the C11A protein and 4-VDH on the one hand and heme on the other is not known. Possible reasons include precipitation of the incorrect rotational isomer b-type cytochrome, which then did not equilibrate with its reactive rotational isomer, or underestimation of the yield because of the problem of estimating the extinction coefficient for the novel cytochromes with single thioether bonds.Fig. 5SDS/15% PAGE of in vitro-formed cytochrome variants of H. thermophilus c552 following incubation with mono-vinyl heme derivatives.A, activity staining for covalently bound heme followed by (B) Coomassie Blue staining. Lane 1, apoform of wild-type (CXXCH) H. thermophilus cytochrome c552. Lane 2, wild-type apoprotein reacted with 2-VDH. Lane 3, wild-type apoprotein reacted with 4-VDH. Lane 4, the C11A mutant of H. thermophilus cytochrome c552 reacted with 2-VDH. Lane 5, the C11A mutant of H. thermophilus cytochrome c552 reacted with 4-VDH. Lane 6, the C14A mutant of H. thermophilus cytochrome c552 reacted with 2-VDH. Lane 7, the C14A mutant of H. thermophilus cytochrome c552 reacted with 4-VDH. All reactions were carried out with 5 μm protein and an equimolar amount of heme or heme derivative, and the reaction time was 15 h. M', ultra-low range protein marker (Sigma) corresponding to 6.5, 14.2, 17.0, and 26.6 kDa from bottom to top, respectively. M, prestained molecular protein marker corresponding nominally to 25 and 16.5 kDa from top to bottom, respectively. 200–400 pmol of protein were applied to each lane of the gel. As noted in the legend to Fig. 2 the relative staining by Coomassie Blue in different lanes is affected by stain retained from the first step of heme staining. Heme stain intensities are not sufficiently quantitative to be used to assess reaction stoichiometries.View Large Image Figure ViewerDownload (PPT) When the C14A mutant of H. thermophilus apocytochrome c552 was used in the reaction with these two heme derivatives, the reactivity was reversed. Again, a b-type cytochrome intermediate was observed on addition of either 2- or 4-VDH (Table I) as was a pyridine hemochrome spectrum with an α-band of 550 nm. For the 4-VDH no spectral change could be observed within 15 h (data not shown), nor was covalent heme attachment detected by SDS-PAGE (Fig. 5, lane 7). However, for the 2-VDH derivative, a spectral change occurred over a period of 15 h (Table I). The resultant pyridine hemochrome spectrum had an α-band of 548 nm. SDS-PAGE analysis confirmed that a reaction had occurred to yield a protein with covalently bound heme (Fig. 5, lane 6). As mentioned earlier, not all the C14A protein bound heme non-covalently. The same was true for binding of mono-vinyl hemes, but in the case of the 2-VDH we estimate, in common with C11A protein and 4-VDH, around 80% of the bound VDH became covalently attached. Overall these data show that the reaction of the cysteine residues with heme vinyl groups is selective to the respective vinyl group and cysteine residue for H. thermophilus cytochrome c552. In the C11A and C14A variants of H. thermophilus cytochrome c552, Cys-11 reacts with the 2-vinyl group of heme and Cys-14 with the 4-vinyl moiety. However, a minor side reaction (up to 10%) leading to non-selective thioether bond formation cannot be excluded as shown by the slight heme staining of the reaction of the C11A mutant with 2-VDH (Fig. 5, lane 4). The kinetics of the reactions of the single cysteine variants of H. thermophilus cytochrome c552 with mono-vinyl heme derivatives were difficult to determine because of the broad absorption bands of the intermediate b-type cytochrome (Fig. 4b for the C11A variant). The broadness of the spectral features of these intermediates might reflect the less rigid heme binding pocket. The lack of the steric hindrance induced by an additional vinyl group of heme might lead to lower constraints on the heme orientations within the protein. Finally, it was shown that wild-type H. thermophilus apocytochrome c552 was able to bind both 2-VDH and 4-VDH covalently (Fig. 5, lanes 2 and 3, respectively), reacting via a b-type cytochrome intermediate (Table I and Fig. 6 show the reaction intermediate and product of wild-type apoprotein with 4-VDH). The apocytochrome was shown to be devoid of covalently bound heme (Fig. 5, lane 1). The kinetics of the monovinyl heme derivatives with wild-type protein containing two cysteine residues were similar to the reaction of heme with apoprotein (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar). However, it again remained unclear whether a biphasic kinetic behavior is observed. C11A and C14A variants of H. thermophilus apocytochrome c552 were shown to attach heme covalently in vitro via thioether bonds. This observation is evidence that the unusual in vivo formation of these cytochrome variants in the cytoplasm of E. coli (8Tomlinson E.J. Ferguson S.J. J. Biol. Chem. 2000; 275: 32530-32534Google Scholar) arises from spontaneous thioether bond formation between heme and apocytochrome. Apoforms of H. thermophilus cytochrome c552 have been shown previously to be relatively compact and to be able to recognize heme rapidly and adopt the characteristics of a b-type cytochrome (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar, 10Tomlinson E.J. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5156-5160Google Scholar, 16Wain R. Pertinhez T.A. Tomlinson E.J. Hong L. Dobson C.M. Ferguson S.J. Smith L.J. J. Biol. Chem. 2001; 276: 45813-45817Google Scholar). We have suggested (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar), but not proved, for wild-type cytochrome c552 of H. thermophilus that the heme may initially bind in two orientations, related to a 180° rotation around the α,γ mesoaxis of heme as has been observed for some other heme proteins following addition in vitro of heme to polypeptide (17La Mar G.N. Toi H. Krishnamoorthi R. J. Am. Chem. Soc. 1984; 106: 6395-6401Google Scholar, 18McLachlan S.J. La Mar G.N. Burns P.D. Smith K.M. Langry K.C. Biochim. Biophys. Acta. 1986; 874: 274-284Google Scholar, 19Yamamoto Y. La Mar G.N. Biochemistry. 1986; 25: 5288-5297Google Scholar, 20Wu J.Z. La Mar G.N. Yu L.P. Lee K.B. Walker F.A. Chiu M.L. Sligar S.G. Biochemistry. 1991; 30: 2156-2165Google Scholar). The wild-type protein, carrying the CXXCH motif, goes on to form a c-type cytochrome under reducing condition in vitro (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar). We argued that this product had the heme covalently attached in only one orientation. The present work provides strong confirmation of this proposal because each of the two mono-vinyl derivatives of heme is selective for the expected single cysteine variant of the cytochrome c, and hence stereoselective product formation is observed with respect to the rotational isomers of the b-type cytochrome complexes relative to the α,γ mesoaxis of heme. It is known that in all naturally formed c-type cytochromes the 2-vinyl group of heme becomes attached to the N-terminal cysteine of the CXXCH motif and the 4-vinyl moiety reacts with the cysteine adjacent to the histidine coordinating to the heme iron (5Barker P.D. Ferguson S.J. Structure. 1999; 7: R281-R290Google Scholar). In general, non-covalently bound heme is found in predominantly one orientation inside proteins such as globins and b-type cytochromes. However, mixtures of heme orientations that are related by a 180° rotation around the α,γ mesoaxis of heme can be seen following addition of heme to an apoprotein in vitro (17La Mar G.N. Toi H. Krishnamoorthi R. J. Am. Chem. Soc. 1984; 106: 6395-6401Google Scholar, 18McLachlan S.J. La Mar G.N. Burns P.D. Smith K.M. Langry K.C. Biochim. Biophys. Acta. 1986; 874: 274-284Google Scholar, 19Yamamoto Y. La Mar G.N. Biochemistry. 1986; 25: 5288-5297Google Scholar). In many cases the apoproteins are thought to present a nascent binding site that can be initially occupied by heme in either orientation. Such binding sites have asymmetric features, which result in one heme orientation being thermodynamically favored. In the case of c-type cytochromes it has not been thought that there is a nascent heme pocket in the apoproteins, but our recent work on both the H. thermophilus cytochrome c552 (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar, 16Wain R. Pertinhez T.A. Tomlinson E.J. Hong L. Dobson C.M. Ferguson S.J. Smith L.J. J. Biol. Chem. 2001; 276: 45813-45817Google Scholar) and developing early studies of others (compare Ref. 21Daltrop O. Ferguson S.J. J. Biol. Chem. 2003; 278: 4404-4409Google Scholar) with mitochondrial cytochrome c (21Daltrop O. Ferguson S.J. J. Biol. Chem. 2003; 278: 4404-4409Google Scholar) challenges this view. The structural features of this putative nascent site are not known, and therefore it may in principle accommodate heme in either of the two rotational isomeric orientations. On the other hand, the heme binding pocket of the apoprotein may have sufficient of the asymmetric features seen in the holoprotein (22Hasegawa J. Yoshida T. Yamazaki T. Sambongi Y. Yu Y. Igarashi Y. Kodama T. Yamazaki K. Kyogoku Y. Kobayashi Y. Biochemistry. 1998; 37: 9641-9649Google Scholar) to ensure preferential binding of heme in one rotational isomeric position. In the former of the two alternatives our results imply that the stereochemical features of the site are such that only a 2- or 4-vinyl group in the same location as in the holoprotein can approach a cysteine thiol sufficiently closely to overcome kinetic constraints on in vitro thioether bond formation. The second alternative implies that the nascent heme binding site in the apoprotein is sufficiently structured so as to exclude its occupancy by heme to yield the “wrong” rotational isomer. In this case bond formation between a mono-vinyl heme and a wrong cysteine can be readily envisaged as not feasible. These considerations also imply that the single thioether bond variants of H. thermophilus cytochromes c formed in the cytoplasm of E. coli (8Tomlinson E.J. Ferguson S.J. J. Biol. Chem. 2000; 275: 32530-32534Google Scholar) have predominantly heme attached in one rotational orientation around the α,γ mesoaxis of heme. This strict stereospecific requirement for covalent attachment of heme to apocytochrome c552 contrasts with the situation seen for uncatalyzed Thermus thermophilus cytochrome c synthesis in the cytoplasm of E. coli, where an inversion of heme relative to its α,γ mesoaxis can be related to misattachment of heme (23McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Google Scholar). This contrast makes the acquisition of structural information about the H. thermophilus apoprotein (16Wain R. Pertinhez T.A. Tomlinson E.J. Hong L. Dobson C.M. Ferguson S.J. Smith L.J. J. Biol. Chem. 2001; 276: 45813-45817Google Scholar) an intriguing prospect. To the best of our knowledge, it is not known what advantage(s) arise from incorporating heme to yield only one rotational isomer in vivo in either c-type cytochromes or into other heme-containing proteins such as globins or b-type cytochromes in a non-covalent fashion. Evolutionary points have been discussed for c-type cytochromes (24Kojo S. Fukunishi K. Tsukamoto I. Angew. Chem. Int. Ed. Engl. 1989; 28: 71-72Google Scholar). We suspect that rotational isomerism arises in vivo either because of stereoselective heme release after its biosynthesis or because hemecontaining proteins have evolved to form the heme pocket such that only one rotational heme-protein isomer yields optimal heme-protein interactions required for efficient functioning of the metalloprotein. Little is known about the chemistry that underpins thioether bond formation from cysteine thiols and vinyl groups of heme. The present observations show, at least for the in vitro studies, that the reaction of either vinyl group of heme to form the first thioether bond is independent of the presence of the second vinyl group. However, where both the second vinyl group and two cysteine residues are present, the formation of the second thioether bond is faster than the first, at least as evidenced by our failure to detect a significant concentration of single thioether bond product during reaction of heme with the CXXCH protein (1Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Google Scholar). The observation that heme lyase can attach heme covalently to apocytochromes with only one cysteine residue in the CXXCH motif (9Rosell F.I. Mauk A.G. Biochemistry. 2002; 41: 7811-7818Google Scholar, 25Tanaka Y. Kubota I. Amachi T. Yoshizumi H. Matsubara H. J. Biochem. 1990; 108: 7-8Google Scholar) suggests that heme lyase might accelerate the process studied here in vitro. Mechanistic implications for the catalytic functions of the heme lyase are discussed elsewhere (21Daltrop O. Ferguson S.J. J. Biol. Chem. 2003; 278: 4404-4409Google Scholar). Overall this work shows that spontaneous formation of a single thioether bond can occur in vitro and can be selective with respect to the heme orientation. It also illustrates that thioether bonds can form independently from one another, i.e. there is no requirement to form both thioether bonds in a concerted fashion in a typical c-type cytochrome. We thank Lin Hong for production of the C11A and C14A mutants of H. thermophilus cytochrome c552." @default.
• W2071434293 created "2016-06-24" @default.
• W2071434293 creator A5001614024 @default.
• W2071434293 creator A5031745695 @default.
• W2071434293 creator A5082370979 @default.
• W2071434293 date "2003-07-01" @default.
• W2071434293 modified "2023-10-03" @default.
• W2071434293 title "Stereoselective in Vitro Formation of c-type Cytochrome Variants from Hydrogenobacter thermophilus Containing Only a Single Thioether Bond" @default.
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