From the
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom and the ¶Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
Received for publication, February 25, 2003 , and in revised form, April 15, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In our previous work (1), we reported the first in vitro formation of a c-type cytochrome containing two thioether bonds. Cytochromes containing only one thioether bond are rare (5). However, engineered variants of the CXXCH binding motif, replacing one cysteine with an alanine residue by site-directed mutagenesis, for a bacterial cytochrome c (8) and for mitochondrial yeast iso-1-cytochrome c (9) 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)1 and 4-vinyldeuteroheme (4-VDH). The latter two molecules are thus hybrids between heme and deuteroheme (Fig. 1).
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SDS-PAGE analysis was carried out using the buffer system described by Laemmli (12); heme activity staining was achieved by the method of Goodhew et al. (13). The concentration of apocytochrome was determined using the extinction coefficient at 280 nm of 15.2 mM1 cm1 (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 (14). 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In Vitro Thioether Bond Formation with HemeMixing 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 -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 (1, 10).
|
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 (3, 8). 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.
|
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 (1). 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 ,
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-VDHTo elucidate whether the reaction of the vinyl groups of heme with the cysteine residues of the apoproteins is selective with respect to the ,
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 (15). 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 -band around 550 nm, which is the average of the pyridine hemochrome maximum of the
-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
-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.
|
|
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 -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
-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 (1). However, it again remained unclear whether a biphasic kinetic behavior is observed.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (1, 10, 16). We have suggested (1), 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 ,
mesoaxis of heme as has been observed for some other heme proteins following addition in vitro of heme to polypeptide (1720). The wild-type protein, carrying the CXXCH motif, goes on to form a c-type cytochrome under reducing condition in vitro (1). 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
,
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 (5).
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 ,
mesoaxis of heme can be seen following addition of heme to an apoprotein in vitro (1719). 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 (1, 16) and developing early studies of others (compare Ref. 21) with mitochondrial cytochrome c (21) 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 (22) 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 (8) have predominantly heme attached in one rotational orientation around the
,
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 ,
mesoaxis can be related to misattachment of heme (23). This contrast makes the acquisition of structural information about the H. thermophilus apoprotein (16) 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 (24). 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 (1).
The observation that heme lyase can attach heme covalently to apocytochromes with only one cysteine residue in the CXXCH motif (9, 25) 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 (21).
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.
![]() |
FOOTNOTES |
---|
Recipient of a University of Oxford scholarship in association with St. Edmund Hall, including a W. R. Miller award.
|| A W. R. Miller Fellow of St. Edmund Hall. To whom correspondence should be addressed. Tel.: 44-0-1865-275240; Fax 44-0-1865-275259; E-mail: stuart.ferguson{at}bioch.ox.ac.uk.
1 The abbreviations used are: 2-VDH, 2-vinyldeuteroheme; 4-VDH, 4-vinyldeuteroheme.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|