Báo cáo khoa học: Structure of a trypanosomatid mitochondrial cytochrome c with heme attached via only one thioether bond and implications for the substrate recognition requirements of heme lyase potx

Structure of a trypanosomatid mitochondrial cytochrome c with heme attached via only one thioether bond and implications for the substrate recognition requirements of heme lyase Vilmos Fu ¨ lo ¨ p 1 , Katharine A. Sam 2 , Stuart J. Ferguson 2 , Michael L. Ginger 3 and James W. A. Allen 2 1 Department of Biological Sciences, University of Warwick, Coventry, UK 2 Department of Biochemistry, University of Oxford, UK 3 School of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, UK The principal physiological role of mitochondrial cyto- chrome c is electron transfer from the cytochrome bc 1 complex to cytochrome aa 3 oxidase during oxidative phosphorylation. c-Type cytochromes form a large family in bacteria, archaea, mitochondria and chlorop- lasts, in which the iron cofactor heme is covalently bound to the polypeptide chain. Such cytochromes have many distinct folds (often unrelated to that of mitochondrial cytochrome c); bacterial c-type cyto- chromes frequently have many hemes [1]. Despite this variety, the covalent heme attachment to protein is highly stereospecific and regiospecific, and is almost Keywords Cytochrome c; heme lyase; intermembrane space; thioether bond; trypanosome Correspondence J. W. A. Allen, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK Fax: +44 0 1865 613201 Tel: +44 0 1865 613330 E-mail: james.allen@bioch.ox.ac.uk Database X-ray structure coordinates for Crithidia fasciculata cytochrome c have been deposited in the Protein Data Bank under the accession code 2w9k (Received 23 December 2008, revised 19 February 2009, accepted 13 March 2009) doi:10.1111/j.1742-4658.2009.07005.x The principal physiological role of mitochondrial cytochrome c is electron transfer during oxidative phosphorylation. c-Type cytochromes are almost always characterized by covalent attachment of heme to protein through two thioether bonds between the heme vinyl groups and the thiols of cyste- ine residues in a Cys-Xxx-Xxx-Cys-His motif. Uniquely, however, members of the evolutionarily divergent protist phylum Euglenozoa, which includes Trypanosoma and Leishmania species, have mitochondrial cytochromes c with heme attached through only one thioether bond [to an (A ⁄ F)XXCH motif]; the implications of this for the cytochrome structures are unclear. Here we present the 1.55 A ˚ resolution X-ray crystal structure of cyto- chrome c from the trypanosomatid Crithidia fasciculata. Despite the funda- mental difference in heme attachment and in the cytochrome c biogenesis machinery of the Euglenozoa, the structure is remarkably similar to that of typical (CXXCH) mitochondrial cytochromes c, both in overall fold and, other than the missing thioether bond, in the details of the heme attach- ment. Notably, this similarity includes the stereochemistry of the covalent heme attachment to the protein. The structure has implications for the maturation of c-type cytochromes in the Euglenozoa; it also hints at a dis- tinctive redox environment in the mitochondrial intermembrane space of trypanosomes. Surprisingly, Saccharomyces cerevisiae cytochrome c heme lyase (the yeast cytochrome c biogenesis system) cannot efficiently mature Trypanosoma brucei cytochrome c or a CXXCH variant when expressed in the cytoplasm of Escherichia coli, despite their great structural similarity to yeast cytochrome c, suggesting that heme lyase requires specific recognition features in the apocytochrome. Abbreviations Ccm, cytochrome c maturation; IMS, intermembrane space; NCS, noncrystallographic symmetry; SHAM, salicylhydroxamic acid. 2822 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS always through two thioether bonds between the vinyl groups of heme and the thiols of cysteine residues that occur in a CXXCH amino acid motif; the histidine serves as the proximal ligand to the heme iron atom [2–4]. However, in one group of eukaryotes, the mem- bers of the protist phylum Euglenozoa, biochemical, spectroscopic and genetic evidence suggests that, uniquely, the mitochondrial c-type cytochromes (c and c 1 ) both have heme covalently bound to the polypeptide chain through only a single thioether bond [to an (F ⁄ A)XXCH motif] [5–10]. The Euglenozoa include ubiquitous, free-living phagotrophic flagellates (e.g. Bodo saltans), photosynthetic algae (e.g. Euglena graci- lis), and parasitic trypanosomatids [e.g. the causal agents of the tropical diseases African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania species)]. Note that some euglenozoans, such as E. gracilis, also contain a chloroplast with typical CXXCH cytochromes c [11]. Covalent attachment of heme to cytochromes c is a catalyzed post-translational modification. Remarkably, at least five completely distinct biogenesis systems have evolved to achieve this heme attachment in different organisms and organelles. Some eukaryotes (land plants and various protists) use the multicomponent System I [also called the cytochrome c maturation (Ccm) system], and others (animals, fungi, and many evolutionarily diverse protozoa and algae) use System III, the enzyme heme lyase, for maturation of their mitochondrial cytochromes c [10]. Surprisingly little is known about the mechanism and substrate recognition features of heme lyase-dependent heme attachment to apocytochrome c, and the origins of this critical eukaryote-specific enzyme are obscure [10]. Strikingly, none of the known cytochrome c biogenesis proteins are present in the several trypanosomatid species for which complete genome sequences are available [9,10,12]. The presence throughout the Euglenozoa of unique single cysteine mitochondrial cytochromes c, coupled with the absence from trypanosomatids of any known cytochrome c biogenesis proteins, points towards a novel maturation apparatus for all eugleno- zoan mitochondrial cytochromes c [9,10]. The reason for the loss of one thioether bond from the mitochondrial cytochromes c of euglenozoans is a longstanding puzzle. It could be a means of altering the structure of these cytochromes c and ⁄ or a conse- quence of some biosynthetic demand. A high-resolu- tion structural comparison between a euglenozoan mitochondrial cytochrome c and a typical cyto- chrome c (with heme bound by two thioether bonds) is therefore important. Moreover, in all the available, diverse structures of c-type cytochromes, there is an invariant stereospecific arrangement of the heme [1,13]; there is no a priori reason to expect this to be the same in the single thioether bond euglenozoan cytochromes. Finally, it is likely that (at least for cytochrome c bio- genesis Systems I and II) folding of holocytochromes c mainly takes place after covalent attachment of heme to the polypeptide chain. Thus, it is not axiom- atic that anchoring the heme to protein through only a single thioether bond would result in the same local structure that is characteristic of heme attachment to a CXXCH motif. Therefore, we have determined the X-ray crystal structure of mitochondrial cytochrome c from the trypanosomatid C. fasciculata; we have also investigated the maturation of trypanosome cyto- chrome c by the poorly understood yeast cytochrome c heme lyase. Results Cytochrome-dependent respiration in C. fasciculata For determination of a euglenozoan cytochrome c structure, we isolated holocytochrome from C. fascicu- lata, a monogenetic insect parasite that is not patho- genic to humans. Some trypanosomatids (e.g. Leishmania spp. and T. cruzi) possess a classic mito- chondrial respiratory chain [14–16]. In others (e.g. the life cycle stage of T. brucei found in the midgut of the tsetse fly), the mitochondrial respiratory chain is branched, and electrons can be transferred from ubiqu- inol to either the cytochrome bc 1 complex, or to the enzyme alternative oxidase, which reduces oxygen to water but is not coupled to ATP production by oxida- tive phosphorylation because proton translocation is absent [17,18]. In other cases, such as pathogenic forms of T. brucei found in the mammalian blood- stream, cytochrome-dependent respiration is repressed, and alternative oxidase is the essential sole terminal oxidase in mitochondrial electron transport [16,19]. Thus, before solving the X-ray structure of C. fascicu- lata cytochrome c, we first confirmed its importance in mitochondrial electron transport in that organism. The presence of 200 lm salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, had little effect on the growth rate of C. fasciculata when compared with control cultures. Addition of 2 lgÆmL )1 antimycin A (a cytochrome bc 1 complex inhibitor), however, resulted in no growth over 72 h (from a starting inocu- lum of 10 5 cellsÆmL )1 ). Similarly, addition of SHAM to a concentration of 3 mm exerted no effect on oxygen consumption by C. fasciculata as measured using a Clark oxygen electrode, whereas following V. Fu ¨ lo ¨ p et al. Structure of Crithidia fasciculata cytochrome c FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2823 addition of antimycin to 2 lgÆmL )1 , oxygen consump- tion by C. fasciculata effectively ceased within a few seconds (Fig. 1). A very similar result was obtained if 2mm KCN was added instead of antimycin A; cyanide inhibits the cytochrome aa 3 oxidase of the classic respiratory chain, but not alternative oxidase. Thus, cytochrome-dependent respiration is essential in C. fasciculata. The structure of C. fasciculata mitochondrial cytochrome c The overall structure of oxidized C. fasciculata cyto- chrome c, determined by X-ray crystallography to a resolution of 1.55 A ˚ , is shown in Fig. 2. The asymmet- ric unit is a trimer. The structure is ordered for resi- dues 5–114 of the 114 amino acid polypeptide chain. The fold is typical for a class I c-type cytochrome (e.g. mitochondrial cytochrome c and bacterial cyto- chromes c 2 ) [20]. The structure unequivocally confirms the earlier conclusion that trypanosomatid cyto- chromes c contain only one thioether bond between heme and protein; there are no other compensatory covalent bonds to the heme cofactor. The heme is (as expected [21]) covalently attached to the protein through its (original) 4-vinyl group (also called the C 8 vinyl), as is observed for the C-terminal cysteine of the typical CXXCH c-type cytochrome heme-binding motif [1,13]. The heme iron is coordinated by the N e of the histidine of the ‘AXXCH’ (actually AAQCH) heme-binding motif, and the sulfur of Met91. The heme iron–ligand distances were restrained to 2 A ˚ (his- tidine) and 2.3 A ˚ (methionine), and the model fits well with these values. The vinyl a-carbon–Cys28 distances were restrained to 1.8 A ˚ , but these refined to consider- ably longer bond lengths: 2.25, 1.98 and 2.00 A ˚ , respectively, for the three subunits of the asymmetric unit. Residue 83 is a trimethyllysine [5]; a moderate fit to the electron density suggests that the trimethyl group is flexible. The structure of C. fasciculata cytochrome c (in red) is overlaid with that of S. cerevisiae iso-1-cytochrome c (in blue) in Fig. 3. The two cytochromes have 48% amino acid identity and 72% similarity. The structures are remarkably similar overall, and in the details of the heme attachment and heme position. The rmsd between the structure of C. fasciculata cytochrome c and S. cerevisiae iso-1-cytochrome c is 0.94 A ˚ for the 104 a-carbon atoms fitted. The positions and stereo- chemistries of both axial heme ligands are essentially Time (s) 0 100 200 300 400 500 + SHAM + Antimycin 55 nmol O 2 Fig. 1. Oxygen consumption by C. fasciculata in the presence of respiratory inhibitors. C. fasciculata cells (3–7 · 10 7 cells in 0.66 mL of culture medium taken directly from a growing culture and diluted to 2 mL with water) were placed in the chamber of a Clark oxygen electrode, and the oxygen consumption of the cells was measured at room temperature. Inhibitors of alternative oxidase (SHAM) and the cytochrome bc 1 complex (antimycin A) were added at the points indicated to final concentrations of 3 m M and 2 lgÆmL )1 , respectively. The electrode was calibrated using a saturated solu- tion of air in water, and water treated with disodium dithionite to remove all of the oxygen. One representative experiment (of seven) is shown. Fig. 2. The X-ray crystal structure of C. fasciculata cytochrome c to 1.55 A ˚ resolution. The molecule is shown rainbow colored, from the N-terminus (residue 5) in blue to the C-terminus (residue 114) in red. The heme cofactor is shown in ball and stick representation, as are the methionine and histidine side chains that coordinate the heme iron, and the cysteine side chain that forms a thioether bond between heme and protein. Also shown is the methyl group of the alanine of the AXXCH heme-binding motif, which is found in place of the first cysteine of a typical c-type cytochrome CXXCH heme- binding motif. Structure of Crithidia fasciculata cytochrome c V. Fu ¨ lo ¨ p et al. 2824 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS identical between the two structures. These observa- tions concur with those of Wuthrich et al., who used proton NMR to investigate the heme environment in cytochrome c from another trypanosomatid species in the 1970s, and concluded that the heme crevice and heme electronic structure were very similar to those in mammalian (CXXCH) mitochondrial cytochrome c [21,22]. (Notably, these authors also correctly predicted the overall structure of the trypanosomatid cyto- chrome.) The stereochemistry of heme attachment through the thioether bond is conserved in C. fascicu- lata cytochrome c (Figs 3 and 4); it therefore remains the same (i.e. S-stereochemistry) as in all known c-type cytochrome structures [1,13]. Strikingly, the methyl group of Ala25 in C. fasciculata cytochrome c (the equivalent residue of the first cysteine of the CXXCH motif of S. cerevisiae cytochrome c) overlays almost perfectly with the CH 2 group of the equivalent cyste- ine. Thus, the position of the polypeptide chain around the cysteine(s) is very similar in the two structures, even though one has attachment through two thioether bonds and the other through only a single bond. As discussed in a recent review [23], ‘the backbone struc- ture of the CXXCH motif [of typical c-type cyto- chromes] shows little variation, even among proteins in which the variable ‘‘XX’’ residues have very different properties’. The observation that this remains true in natural single thioether cytochrome c is a notable finding. Details of the heme attachment in C. fasciculata cytochrome c are shown in Fig. 4. The methyl group Fig. 3. Comparison between the structures of C. fasciculata cyto- chrome c (protein main chain in red) and S. cerevisiae iso-1-cyto- chrome c (Protein Data Bank entry: 1YCC) (in blue). Also shown are the heme ligands (histidine and methionine in each case), the cysteines that form thioether bonds to the heme, and the methyl group of the alanine of the C. fasciculata AXXCH heme-binding motif, which is found in place of the first cysteine of the S. cerevi- siae CXXCH heme-binding motif. The rmsd between the structure of C. fasciculata cytochrome c and S. cerevisiae iso-1-cytochrome c is 0.94 A ˚ for the 104 a-carbon atoms fitted. Fig. 4. Detail of the heme-binding site in C. fasciculata cyto- chrome c from two angles. The SIGMAA [50] weighted 2mF o )DF c electron density, using phases from the final model of the half- reduced form, is contoured at the 1.5r level, where r represents the rms electron density for the unit cell. Contours more than 1.5 A ˚ from any of the displayed atoms have been removed for clarity. Thin lines indicate heme axial ligand coordination and hydrogen bonds. Sulfur atoms (in methionine and cysteine) are colored yellow, nitrogen blue, oxygen red, and iron purple. The methyl group of the alanine of the AXXCH heme-binding motif is green, and the unsaturated vinyl group of heme is cyan. Pro41 is conserved in class I c-type cyto- chromes, and its main chain carbonyl is hydrogen bonded to the N d atom of the heme axial histidine side chain; this interaction main- tains the correct orientation of the histidine ring to the heme iron. V. Fu ¨ lo ¨ p et al. Structure of Crithidia fasciculata cytochrome c FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2825 of the alanine of the AXXCH motif (Ala25) (in green) and the unsaturated vinyl group of the heme (cyan) are separated by 3.41 A ˚ (as compared with a typical thioether bond length of 1.8 A ˚ ). As the posi- tion of the polypeptide chains in this region is essen- tially the same in the two structures (Fig. 3), this means that in the C. fasciculata cytochrome the heme moves away from the polypeptide relative to its position in the S. cerevisiae protein. The unsatu- rated 2-vinyl group of C. fasciculata cytochrome c remains almost coplanar with the porphyrin ring. The conserved residue Pro41 is hydrogen bonded through its carbonyl group to the N d of the proxi- mal heme histidine ligand [20]. The electron density shows no additional modifications (e.g. oxidation) to the sulfurs of the heme-binding methionine or cyste- ine residues. Maturation of trypanosome cytochrome c by yeast cytochrome c heme lyase Given the remarkable overall structural similarity between the mitochondrial cytochromes c from C. fas- ciculata and S. cerevisiae (Fig. 3), we also investigated whether the poorly understood enzyme responsible for heme attachment to cytochrome c in yeast, heme lyase, can mature a trypanosomatid cytochrome c. Heme lyases can mature AXXCH variants of yeast or human cytochrome c, albeit at a lower level than the CXXCH wild-type [24,25]. Thus, we coexpressed S. cerevisiae cytochrome c heme lyase with either T. brucei cyto- chrome c or a CXXCH variant in the cytoplasm of E. coli (the cytochromes c from C. fasciculata and T. brucei have 84% sequence identity and 92% simi- larity, and both have an AAQCH heme-binding motif). As a control, we also coexpressed heme lyase with S. cerevisiae iso-1-cytochrome c. Cells expressing the yeast cytochrome were bright red, and were shown by absorption spectroscopy to have produced $ 1.4 mg of cytochrome c per gram of wet cells (assuming a reduced Soret band extinction coefficient of 130 000 m )1 Æcm )1 [20]). However, neither wild-type T. brucei cytochrome c nor the CXXCH variant was matured at levels immediately detectable by spectros- copy or by staining of SDS ⁄ PAGE gels for proteins with covalently bound heme. Expression of the protein was, however, readily confirmed by western blotting of the E. coli cytoplasmic extracts using a polyclonal antibody raised against recombinant T. brucei CXXCH holocytochrome c (Fig. 5A), the latter matured by the E. coli Ccm system [9]; this antibody is sensitive to both T. brucei holocytochrome c and T. brucei apocytochrome c (J. W. A. Allen, unpub- lished observation). Following concentration of the E. coli cytoplasmic extracts, both T. brucei wild-type holocytochrome c and T. brucei CXXCH holocyto- chrome c could be observed on heme-stained SDS ⁄ PAGE gels, and ran at the same molecular mass as purified recombinant T. brucei CXXCH cytochrome c matured by the E. coli Ccm system A B 14 kDa 14 kDa 400 450 500 550 600 0.00 0.02 0.04 0.06 0.08 0.10 C 1 2 3 4 1 2 3 4 5 6 7 8 9 10 Normalised absorbance Wavelength (nm) Fig. 5. Maturation of T. brucei cytochrome c and a CXXCH variant by S. cerevisiae cytochrome c heme lyase. (A) Western blot of cytoplasmic extracts from E. coli coexpressing heme lyase and cytochrome c, using a primary antibody raised against the CXXCH variant of T. brucei holocytochrome c (the latter matured in the periplasm of E. coli by the E. coli Ccm apparatus [9]). Lane 1: molecular mass markers. Lanes 2–5: four independent cultures expressing wild-type T. brucei cytochrome c. Lanes 6–9: four inde- pendent cultures expressing the CXXCH variant of T. brucei cyto- chrome c. Lane 10: as the positive control, purified CXXCH variant T. brucei holocytochrome c matured in the periplasm of E. coli by the E. coli Ccm apparatus. (B) SDS ⁄ PAGE gel of concentrated cyto- plasmic extracts from E. coli coexpressing heme lyase and cyto- chrome c, stained for proteins containing covalently bound heme. Lane 1: molecular mass markers. Lane 2: wild-type T. brucei cyto- chrome c. Lane 3: the CXXCH variant of T. brucei cytochrome c. Lane 4: as the positive control, purified CXXCH variant T. brucei holocytochrome c matured in the periplasm of E. coli by the E. coli Ccm apparatus. (C) Absorption spectra of concentrated cytoplasmic extracts from E. coli coexpressing heme lyase and cytochrome c. Wild-type T. brucei cytochrome c (black line) and the CXXCH variant (gray line). A few grains of disodium dithionite were added to the samples to reduce the cytochromes. As no extinction coefficients are available, the spectra were normalized by intensity of the Soret band. The spectra were also corrected for light scattering by sub- traction of a wavelength to the power four curve. Structure of Crithidia fasciculata cytochrome c V. Fu ¨ lo ¨ p et al. 2826 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS (Fig. 5B). Yields of heme lyase-matured T. brucei holocytochrome were calculated from absorption spectra of the concentrated cytochromes (Fig. 5C) as 3.1 lg (wild-type) and 3.2 lg (CXXCH) of holocyto- chrome c per gram of wet cells, assuming in each case a reduced Soret band extinction coefficient of 130 000 m )1 Æcm )1 . This cytochrome maturation was heme lyase-dependent, because virtually no holocyto- chrome was observed if expressed in the absence of heme lyase. We previously investigated maturation of T. brucei cytochrome c in E. coli by the Ccm system, which produced approximately 1.6 mg of CXXCH variant holocytochrome per gram of wet cells [9]; hence, the low yields of heme lyase-matured holo- cytochrome in the present work are not due to, for example, poor expression of the apoprotein or T. brucei codon usage. We conclude that both wild- type and CXXCH T. brucei cytochrome c are matured by heme lyase, to similar extents, but, sur- prisingly, at only approximately 0.25% of the level of maturation of S. cerevisiae iso-1-cytochrome c, even though the various cytochromes are structurally extremely similar. Discussion Euglenozoan cytochrome c – evolution and maturation We report here the first high-resolution structure of mitochondrial cytochrome c from a euglenozoan organism. The mitochondrial cytochromes c and c 1 from this evolutionarily divergent protist group are unique because they contain heme covalently bound through only one cysteine residue to an (A ⁄ F)XXCH heme-binding motif, rather than through two thioether bonds to CXXCH, as in all other eukaryotes. No apparatus for the post-translational attachment of heme to apocytochrome c has yet been identified in any euglenozoan, and, in contrast to all other eukary- otes possessing mitochondrial cytochromes c, no appa- ratus is evident from the analysis of multiple completely sequenced trypanosomatid genomes [9,10,12]. Identification of the novel biogenesis system for cytochrome c in trypanosomes is a demanding task. Remarkably, despite these fundamental differ- ences in heme attachment and cytochrome biogenesis, the structure of C. fasciculata cytochrome c is very similar to the structures of typical mitochondrial cyto- chromes c, e.g. from S. cerevisiae (Fig. 3). This simi- larity was also observed, other than the missing thioether bond, in the details of the heme attachment and around the heme-binding site (Figs 3 and 4) [21]. Different c-type cytochromes have very different folds [1], but the structural arrangement of the heme-bind- ing motif around the thioether linkages is absolutely conserved. The present work extends this observation to a cytochrome with a natural single cysteine heme- binding motif. Moreover, as illustrated with E. gracilis cytochrome c 558 , single thioether attachment of heme does not significantly affect the reaction between the cytochrome c and (mammalian) cytochrome bc 1 or cytochrome aa 3 oxidase (when compared with horse heart cytochrome c) [26]. The biophysical properties of euglenozoan and typical mitochondrial cyto- chromes c are also similar [8]. Priest and Hajduk [6] speculated that single thioether heme attachment in both cytochromes c and c 1 (rather than just in one) might be mutually compensatory, allowing efficient interaction between them for respiratory electron transfer in spite of their distinctive mode of heme binding. However, our data (Fig. 3) suggest that the unique mode of heme attachment is probably unre- lated to the interaction between cytochrome c and its redox partners. There is no apparent structural com- pensation for the presence of only one thioether bond in C. fasciculata cytochrome c, although properties such as the reduction potential may be subtly fine- tuned by the protein. There is no obvious reason from the protein struc- ture or functional data why one group of protists has evolved a unique type of cytochrome c and a corre- sponding novel biogenesis pathway. However, it is clear from our data that euglenozoan cytochrome c could structurally accommodate two cysteines in a typical CXXCH heme-binding motif (Fig. 3). So, how might the occurrence of these single cysteine cyto- chromes c be explained? Considerable evidence points to catalyzed formation and subsequent reduction of an intramolecular disulfide bond in the CXXCH motif during cytochrome c biogenesis in bacteria [3]; this may also happen in yeast [27]. It therefore seems plausible that evolution of the euglenozoan single cys- teine heme-binding motif, while the protein structure was otherwise retained (Fig. 3), relates to the redox environment of the euglenozoan mitochondrial inter- membrane space (IMS) (the location of the cyto- chrome c). Loss of one cysteine from the cytochrome c heme-binding motif could: (a) signifi- cantly affect the interactions between the apocyto- chrome and other thiol proteins in the IMS; and ⁄ or (b) prevent the formation of an undesirable intramo- lecular disulfide bond in the apocytochrome for which no suitable reductant would be available in the IMS; and ⁄ or (c) provide a selective advantage by alle- viating a constraint on other IMS redox proteins. V. Fu ¨ lo ¨ p et al. Structure of Crithidia fasciculata cytochrome c FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2827 Our structure thus adds to other recent evidence [28] hinting that the redox environment of the mitochon- drial IMS in trypanosomatids may be different from that in animals and yeast. Notably, the stereochemistry of heme attachment remains conserved in all cytochromes c, including that from C. fasciculata (Figs 3 and 4). Heme is not symmetrical; to date, it has always been observed to be attached to cytochromes c with S-stereochemistry [1], although the physiological advantage of such ste- reospecific attachment is not known. It was therefore not clear a priori whether euglenozoan cytochromes c with heme attached through a single cysteine would have the same attachment stereochemistry as cyto- chromes c with two thioether linkages [13]. The fact that they do must be reflected in stereospecific con- trol of the heme attachment by the euglenozoan cyto- chrome c biogenesis machinery. If heme is not inserted into cytochromes in a controlled orientation, it enters initially in a mixed, roughly equal popula- tion, and equilibrates slowly until one orientation dominates (as observed for b-type cytochromes and globins) [29–31]. This implies that there may be a heme-handling chaperone in the trypanosomatid cytochrome c biogenesis machinery (as there is in biogenesis System I [32]). One surprising difference in the structure of C. fas- ciculata cytochrome c relative to ‘normal’ CXXCH c-type cytochromes is that the thioether bond between heme and protein is longer than is typical (1.98– 2.25 A ˚ as compared with 1.8 A ˚ ). Our structure was refined with a nonredundant first part of the dataset, which showed similar bond lengths, as did refinement against a lower-resolution dataset collected at a much less intense beamline (ESRF, BM16). Therefore, this observation cannot be interpreted as a result of X-ray-induced radiation damage; rather, it is an intrinsic feature of the structure. In single thioether cytochrome c, the heme is less constrained than in a normal (CXXCH) c-type cytochrome, because it is covalently anchored to the protein only once rather than twice. This leads to greater conformational flexi- bility of the heme, which is reflected, for example, in broadening of the peaks in the absorption spectrum [9,24]. Moreover, when heme is attached to a CXXCH motif, the (quite significant) strain of con- straining the heme position is spread over two thioe- ther bonds plus the histidine ligand to the iron, whereas in the euglenozoan mitochondrial cyto- chromes, the load must be borne by only one thioe- ther bond plus the histidine. Together, these factors presumably lead to a weaker, and hence longer, thioe- ther bond. Cytochrome c maturation by other biogenesis systems The structure reported here is also informative in the context of the failure of the E. coli Ccm apparatus to effectively mature wild-type (AXXCH) T. brucei cyto- chrome c [9]; the system can mature both a CXXCH variant [9] and the structurally very similar (Fig. 3) yeast (CXXCH) mitochondrial cytochrome c [33]. Hence, we can now conclude with a high degree of confidence that the inability of the Ccm system to mature the single cysteine trypanosomatid cyto- chrome c was due to the Ccm apparatus itself, and not to some (previously unidentified) structural feature of the substrate cytochrome. It has been argued that cyto- chrome c biogenesis System II can catalyze single cysteine heme attachment within the four-heme c-type cytochrome NrfH from Wolinella succinogenes which is unrelated to mitochondrial cytochrome c [34]. The present work suggests that such single cysteine attach- ment could be structurally accommodated within that protein as a variant of the wild-type double cysteine attachment. The bioinformatic implications are clear; an XXXCH sequence could be indicative of a c-type cytochrome in genomes of bacterial species that use System II [34]. (Note also that the cytochrome b 6 f complex of cyanobacteria and chloroplasts contains a heme covalently bound to protein via a single thioether bond; this heme attachment is dependent on the recently described biogenesis System IV [12].) Our data further show, unexpectedly, that S. cerevi- siae heme lyase (System III) matures a CXXCH vari- ant of T. brucei cytochrome c very poorly, in spite of the great structural homology between the trypanoso- matid and yeast cytochromes. There are two extreme possibilities for how the scarcely understood heme lyase recognizes its target apocytochrome. The first, by analogy with the Ccm system [35], is that it recognizes little more than the CXXCH heme-binding motif. The second is that it recognizes as yet undefined features of the apoprotein, leading to a productive complex within which heme is attached. Our results here suggest the second possibility, and that the recognition features in the apocytochrome are not related to the overall struc- ture of the cytochrome. This complements the previous observation that heme lyase is unable to mature a bacterial class I c-type cytochrome, Paracoccus denitrif- icans cytochrome c 550 [33]. Moreover, many taxa that have heme lyase apparently have separate heme lyases for the maturation of cytochromes c and c 1 [10]; this has been demonstrated biochemically for S. cerevisiae, where only very limited overlap of substrate specificity was observed [36]. Again, ‘simple’ interaction between Structure of Crithidia fasciculata cytochrome c V. Fu ¨ lo ¨ p et al. 2828 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS heme lyase and the apocytochrome CXXCH motif would appear to be unlikely if separate heme lyases are required to mature cytochromes c and c 1 . Bernard et al. [36] identified point mutations in S. cerevisiae cyto- chrome c 1 that enhanced the activity of cytochrome c heme lyase towards the former protein. The identifica- tion of such residues both within and upstream of the CXXCH heme-binding motif supports our conclusion that heme lyase recognizes specific features in its apocy- tochrome substrate, rather than just the heme-binding motif or the overall fold of the protein. Experimental procedures C. fasciculata choanomastigotes were cultured at 27 °Cin media containing 37 gÆL )1 heart–brain infusion, 10 mgÆL )1 hemin, 10 mgÆL )1 folic acid and 5% (v ⁄ v) heat-inactivated fetal bovine serum. Cells inoculated at $ 10 4 cellsÆmL )1 in 500 mL tissue culture flasks containing 100 mL of medium were grown for 60–72 h. Harvested cells were washed twice in NaCl ⁄ P i (8 gÆL )1 NaCl, 0.2 gÆL )1 KCl, 1.42 gÆL )1 Na 2 HPO 4 and 0.27 gÆL )1 KH 2 PO 4 , pH 7.2) and stored at )80 °C until required. Growth assays for C. fasciculata were conducted by the addition of respiratory inhibitors as described in the text; growth was assessed either by counts using a hemocytometer, or by measurement of D 600 nm val- ues. Respiration of C. fasciculata was also investigated using an oxygen electrode (Rank Brothers, Bottisham, UK), which was calibrated and used according to the man- ufacturer’s directions. Cells were placed in the electrode chamber in their growth medium, and respiratory inhibitors were added as required. Purification of cytochrome c Extracts of C. fasciculata were prepared by disrupting the cells from $ 20 L of culture twice in buffer containing 1.42 gÆL )1 Na 2 HPO 4 , 0.27 gÆL )1 KH 2 PO 4 ,1mm Na 2 EDTA, 2 mm EGTA, 5 lm 2-mercaptoethanol, 0.25 mm phenylmethanesulfonyl fluoride, four ‘Complete’ prote- ase inhibitor tablets (Roche) for every 50 mL of buffer (containing $ 2 · 10 11 cells), and 2% (v ⁄ v) Nonidet P40 Substitute detergent (Igepal CA-630; USB Corporation, Cleveland, OH, USA). Cell debris was removed by centrifu- gation at 17 000 g for 15 mins, and the soluble cell extracts were diluted five-fold with 50 mm Tris ⁄ HCl buffer (pH 8.0), and then applied to an XK26 ⁄ 20 column containing SP-Sepharose fast-flow resin (GE Healthcare, Amersham, UK) at room temperature. The column was washed with the same buffer, and then with 50 mL of 2 mm K 3 Fe(CN) 6 dissolved in the buffer to ensure that the C. fasciculata cytochrome c (sometimes called cytochrome c 555 [5]) was all oxidized. The protein was eluted from the column with a 500 mL gradient of 0–500 mm NaCl in 50 mm Tris ⁄ HCl buffer (pH 8.0), with a flow rate of 10 mLÆmin )1 ;8mL fractions were collected. Fractions were assessed by their red color, and those with maximum Soret band absorbance more than one-third that of the best fraction were retained. The pooled fractions were diluted five-fold in 50 m m Tris ⁄ HCl (pH 8.0), and applied to an XK26 ⁄ 20 column containing CM-Sepharose fast-flow resin (GE Healthcare) at room temperature. The protein was eluted as described above. Retained fractions containing the purest cytochrome were concentrated to a volume of $ 1.5 mL, and applied to a Sephacryl S-200 column (2.6 cm diameter, 1 m length), pre-equilibrated with 50 mm potassium phosphate buffer (pH 7.0). This chromatography step was conducted at 4 °C. The cytochrome was eluted in the same buffer at a flow rate of 15 mLÆh )1 ; 5 mL fractions were collected and assessed for purity by absorption spectroscopy and SDS ⁄ PAGE. Those with A Soret (oxidized) ⁄ A 280 nm (oxi- dized) > 3.8 were regarded as pure, and were concentrated to 16 mgÆmL )1 ($ 1.3 mm) for crystallography. Crystallization, structure determination, and model refinement Redox homogeneity of the purified C. fasciculata cyto- chrome c was ensured by the addition of 5 mm K 3 Fe(CN) 6 . Crystals initially formed in sitting drops made by mixing the protein 1 : 1 with a solution containing 2.7 m (NH 4 ) 2 SO 4 , 0.1 m Hepes (pH 6.5) and 0.1 m LiCl at 19 °C (all crystallographic reagents purchased from Hampton Research, Aliso Viejo, CA, USA). These crystals were of poor quality, but they were used for seeding. Diffraction- quality crystals were produced in hanging drops by mixing the protein 1 : 1 with 2.45 m (NH 4 ) 2 SO 4 and 0.1 m Hepes (pH 6.5); these drops were seeded with microcrystals after equilibration for $ 48 h. Crystals grew, and were harvested, within 1 week. Crystals were then picked up from the mother liquor containing 15% glycerol using a cryoloop, placed in a nitrogen stream at 100 K, and stored in liquid nitrogen until data collection. Initial diffraction data were collected at beamline BM16 (European Synchrotron Radia- tion Facility), but the final dataset used for structure deter- mination and refinement was collected at the Diamond Light Source, UK. Integration and scaling were performed using denzo and scalepack [37]. Subsequent data handling was carried out using the ccp4 software package [38]. Molecular replacement was carried out using the coordi- nates of S. cerevisiae iso-1-cytochrome c (Protein Data Bank code: 1YCC) as a search model with the phaser pro- gram [39]. Refinement of the structure was carried out by alternate cycles of refmac [40], using noncrystallographic symmetry restraints and manual rebuilding in o [41]. Water molecules were added to the atomic model automatically by arp ⁄ warp [42], and in the last steps of refinement all the noncrystallographic symmetry restraints were released. V. Fu ¨ lo ¨ p et al. Structure of Crithidia fasciculata cytochrome c FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2829 A summary of the data collection and refinement statistics is given in Table 1. Figures were drawn using molscript [43,44] and rendered with raster 3d [45]. Heme lyase maturation assays Maturation of T. brucei cytochrome c by heme lyase was investigated in the cytoplasm of E. coli BL21-DE3 cells. Plasmids for T. brucei cytochrome c (pKK223–Tbcytc), its CXXCH variant (pKK223–TbcytcCXXCH), S. cerevisiae cytochrome c heme lyase (pACcyc3) and iso-1-cytochrome c (pScyc1) were as previously described [9,33]. Cells were cotransformed with the heme lyase plasmid and the plasmid for each of the cytochromes, respectively. Cells were grown overnight at 37 °C with vigorous shaking, in 50 mL of 2· TY medium (16 gÆL )1 peptone, 10 gÆL )1 yeast extract, 5gÆL )1 NaCl) supplemented with 100 lgÆmL )1 ampicillin, 34 lgÆmL )1 chloramphenicol and 1 mm isopropyl-thio-b-d- galactoside. Nine separate cultures were grown for each combination of heme lyase and cytochrome. The E. coli periplasmic fraction was prepared as previously described [46], and discarded. The spheroplast pellet was resuspended by vigorous vortexing in 50 mm Tris ⁄ HCl plus 150 mm NaCl (pH 7.3), and broken by six freeze–thaw cycles (at )78 and 37 °C); this was followed by centrifugation at 25 000 g for 1 h to remove the cell debris. The soluble cyto- plasmic fraction was initially assayed by running the pro- teins on SDS ⁄ PAGE gels that were stained for proteins containing covalently bound heme [47]. Subsequently, the extracts from multiple cultures were pooled and applied to a 5 mL Hi-Trap column containing SP-Sepharose (GE Healthcare). The bound protein was batch eluted using 500 mm NaCl, concentrated, and then assessed using absorption spectroscopy and heme-stained SDS ⁄ PAGE gels. Western blotting was performed using a polyclonal primary antibody raised against purified, recombinant, Ccm system-matured CXXCH variant T. brucei holocyto- chrome c (protein as described in [9]; antibody raised by Covalab, Villeurbanne, France). Unconcentrated E. coli soluble cytoplasmic extracts were resolved by SDS ⁄ PAGE and blotted onto Hybond-C Extra nitrocellulose membrane (GE Healthcare). The membrane was blocked for 1 h in 5% (w ⁄ v) milk powder dissolved in NaCl ⁄ Tris [50 mm Tris ⁄ HCl, pH 7.5, 120 mm NaCl, 1% (v ⁄ v) Tween-20]. It was then incubated for 1 h with primary antibody diluted 200-fold in 10 mL of 5% milk ⁄ NaCl ⁄ Tris solution; the primary anti- body was used as crude (unpurified) serum. The membrane was washed four times (1 · 15 min, 3 · 5 min) in 10 mL of NaCl ⁄ Tris, and then incubated with the secondary antibody for 1 h in 30 mL of 5% milk ⁄ NaCl ⁄ Tris; the secondary anti- body was affinity purified anti-rabbit IgG whole molecule alkaline phosphatase conjugate (purchased from Sigma, Poole, UK), and was used at 6000-fold dilution. The mem- brane was then washed three times in NaCl ⁄ Tris (each wash for 5 min), and stained by incubation in 10 mL of H 2 O containing a dissolved FAST 5-bromo-4-chloroindol-2-yl phosphate ⁄ Nitro Blue tetrazolium tablet (Sigma). Acknowledgements This work was supported by the Biotechnology and Biological Sciences Research Council [grant numbers BB ⁄ C508118 ⁄ 1 and BB ⁄ D019753 ⁄ 1]. J. W. A. Allen is a BBSRC David Phillips Fellow, and M. L. Ginger is a Royal Society University Research Fellow. KAS is the William R. Miller Junior Research Fellow, Table 1. Summary of crystallographic data collection and refine- ment statistics. Numbers in parentheses refer to values in the high- est-resolution shell. R sym ¼ P j P h I h;j ÀhI h ij   . P j P h hI h i, where I h,j is the jth observation of reflection h, and <I h > is the mean inten- sity of that reflection. R cryst ¼ P F obs jj À F calc jjjj= P F obs j , where F obs and F calc are the observed and calculated structure factor amplitudes, respectively. R free is equivalent to R cryst for a 4% sub- set of reflections not used in the refinement [48]. DPI, diffraction component precision index [49]. Data collection Synchrotron radiation, detector and wavelength (A ˚ ) Diamond, IO2, ADSC Q315 CCD 0.9511 Unit cell (A ˚ ) a = 85.93, b = 110.88, c = 60.93, b = 131.2 Space group C2 Resolution (A ˚ ) 56–1.55 (1.61–1.55) Observations 392 320 Unique reflections 60 049 I ⁄ r(I) 14.8 (2.0) R sym 0.118 (0.693) Completeness (%) 97.6 (100.0) Refinement Nonhydrogen atoms 3199 (including three c-type hemes, seven sulfates and 563 waters) R cryst 0.210 (0.291) Reflections used 57 638 (4079) R free 0.247 (0.320) Reflections used 2411 (171) R cryst (all data) 0.212 Average temperature factor (A ˚ 2 ) 24.4 Protein 21.3 Hemes 15.4 Solvent 39.4 Wilson plot 21.6 Rmsds from ideal values Bonds (A ˚ ) 0.016 Angles (°) 1.7 DPI coordinate error (A ˚ ) 0.09 Ramachandran plot Most favored (%) 89.1 Additionally allowed (%) 10.9 Structure of Crithidia fasciculata cytochrome c V. Fu ¨ lo ¨ p et al. 2830 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS St Edmund Hall, Oxford. We thank N. Brown for very helpful crystallographic discussions, A. Holehouse for preliminary experiments, and H. Lill for the gift of plasmids encoding the S. cerevisiae proteins used in this work. Crystallographic data were collected at beamline IO2 at Diamond Light Source, UK, and we acknowledge the support of T. Sorensen at Diamond and A. Labrador at the ESRF, France. References 1 Barker PD & Ferguson SJ (1999) Still a puzzle: why is haem covalently attached in c-type cytochromes? Struc- ture 7, R281–R290. 2 Stevens JM, Daltrop O, Allen JWA & Ferguson SJ (2004) C-type cytochrome formation; chemical and biological enigmas. Acc Chem Res 37, 999–1007. 3 Allen JWA, Daltrop O, Stevens JM & Ferguson SJ (2003) C-type cytochromes: diverse structures and bio- genesis systems pose evolutionary problems. Philos Trans R Soc Lond B Biol Sci 358, 255–266. 4 Kranz R, Lill R, Goldman B, Bonnard G & Merchant S (1998) Molecular mechanisms of cytochrome c biogenesis: three distinct systems. Mol Microbiol 29, 383–396. 5 Hill GC & Pettigrew GW (1975) Evidence for the amino-acid sequence of Crithidia fasciculata cytochrome c 555 . Eur J Biochem 57, 265–271. 6 Priest JW & Hajduk SL (1992) Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c 1 . J Biol Chem 267, 20188–20195. 7 Pettigrew GW, Leaver JL, Meyer TE & Ryle AP (1975) Purification, properties and amino acid sequence of atypical cytochrome c from two protozoa, Euglena gracilis and Crithidia oncopelti. Biochem J 147, 291–302. 8 Pettigrew GW, Aviram I & Schejter A (1975) Physico- chemical properties of two atypical cytochromes c, Crithidia cytochrome c 557 and Euglena cytochrome c 558 . Biochem J 149, 155–167. 9 Allen JWA, Ginger ML & Ferguson SJ (2004) Matura- tion of the unusual single cysteine (XXXCH) mitochon- drial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway. Biochem J 383, 537–542. 10 Allen JWA, Jackson AP, Rigden DJ, Willis AC, Ferguson SJ, Willis AC, Ferguson SJ & Ginger ML (2008) Order within a mosaic distribution of mitochon- drial c-type cytochrome biogenesis systems? FEBS J 275, 2385–2402. 11 Pettigrew GW (1974) The purification and amino acid sequence of cytochrome c 552 from Euglena gracilis. Bio- chem J 139, 449–459. 12 Kuras R, Saint-Marcoux D, Wollman FA & de Vitry C (2007) A specific c-type cytochrome maturation system is required for oxygenic photosynthesis. Proc Natl Acad Sci USA 104, 9906–9910. 13 Hamel P, Corvest V, Giege P & Bonnard G (2008) Bio- chemical requirements for the maturation of mitochon- drial c-type cytochromes. Biochim Biophys Acta 1793 , 125–138. 14 van Hellemond JJ, Hoek A, Schreur PW, Chupin V, Ozdirekcan S, Geysen D, van Grinsven KW, Koets AP, Van den Bossche P, Geerts S et al. (2007) Energy metabolism of bloodstream form Trypanosoma theileri. Eukaryot Cell 6, 1693–1696. 15 Guerra DG, Decottignies A, Bakker BM & Michels PA (2006) The mitochondrial FAD-dependent glycerol-3- phosphate dehydrogenase of Trypanosomatidae and the glycosomal redox balance of insect stages of Trypanoso- ma brucei and Leishmania spp. Mol Biochem Parasitol 149, 155–169. 16 van Hellemond JJ, Simons B, Millenaar FF & Tielens AG (1998) A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp. J Eukaryot Microbiol 45, 426–430. 17 Chaudhuri M, Ord RD & Hill GC (2006) Trypanosome alternative oxidase: from molecule to function. Trends Parasitol 22, 484–491. 18 Lamour N, Riviere L, Coustou V, Coombs GH, Barrett MP et al. (2005) Proline metabolism in procyclic Try- panosoma brucei is down-regulated in the presence of glucose. J Biol Chem 280, 11902–11910. 19 Helfert S, Estevez AM, Bakker B, Michels P & Clayton C (2001) Roles of triosephosphate isomerase and aero- bic metabolism in Trypanosoma brucei. Biochem J 357, 117–125. 20 Moore GR & Pettigrew GW (1990) Cytochromes c: Evolutionary, Structural, and Physicochemical Aspects . Springer-Verlag, New York, NY. 21 Keller RM, Picot D & Wuthrich K (1979) Individual assignments of the heme resonances in the 360 MHz 1 H NMR spectra of cytochrome c 557 from Crithidia onco- pelti. Biochim Biophys Acta 580, 259–265. 22 Keller RM, Pettigrew GW & Wuthrich K (1973) Struc- tural studies by proton NMR of cytochrome c 557 from Crithidia oncopelti. FEBS Lett 36, 151–156. 23 Bowman SEJ & Bren KL (2008) The chemistry and bio- chemistry of heme c: functional bases for covalent attachment. Nat Prod Rep 25, 1118–1130. 24 Rosell FI & Mauk AG (2002) Spectroscopic properties of a mitochondrial cytochrome c with a single thioether bond to the heme prosthetic group. Biochemistry 41, 7811–7818. 25 Tanaka Y, Kubota I, Amachi T, Yoshizumi H & Matsubara H (1990) Site-directedly mutated human cytochrome c which retains heme c via only one thioether bond. J Biochem 108, 7–8. V. Fu ¨ lo ¨ p et al. Structure of Crithidia fasciculata cytochrome c FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2831 [...]... Hajduk SL (1992) Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1 J Biol Chem 267, 20188–20195 7 Pettigrew GW, Leaver JL, Meyer TE & Ryle AP (1975) Purification, properties and amino acid sequence of atypical cytochrome c from two protozoa, Euglena gracilis and Crithidia oncopelti Biochem J 147, 291–302 8 Pettigrew GW, Aviram I & Schejter A (1975) Physicochemical... Mauk AG (2002) Spectroscopic properties of a mitochondrial cytochrome c with a single thioether bond to the heme prosthetic group Biochemistry 41, 7811–7818 Tanaka Y, Kubota I, Amachi T, Yoshizumi H & Matsubara H (1990) Site-directedly mutated human cytochrome c which retains heme c via only one thioether bond J Biochem 108, 7–8 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation... performed using a polyclonal primary antibody raised against purified, recombinant, Ccm system-matured CXXCH variant T brucei holocytochrome c (protein as described in [9]; antibody raised by Covalab, Villeurbanne, France) Unconcentrated E coli soluble cytoplasmic extracts were resolved by SDS ⁄ PAGE and blotted onto Hybond -C Extra nitrocellulose membrane (GE Healthcare) The membrane was blocked for 1 h in... Structure of Crithidia fasciculata cytochrome c 13 14 References 1 Barker PD & Ferguson SJ (1999) Still a puzzle: why is haem covalently attached in c- type cytochromes? Structure 7, R281–R290 2 Stevens JM, Daltrop O, Allen JWA & Ferguson SJ (2004) C- type cytochrome formation; chemical and biological enigmas Acc Chem Res 37, 999–1007 3 Allen JWA, Daltrop O, Stevens JM & Ferguson SJ (2003) C- type cytochromes:... Physicochemical properties of two atypical cytochromes c, Crithidia cytochrome c5 57 and Euglena cytochrome c5 58 Biochem J 149, 155–167 9 Allen JWA, Ginger ML & Ferguson SJ (2004) Maturation of the unusual single cysteine (XXXCH) mitochondrial c- type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway Biochem J 383, 537–542 10 Allen JWA, Jackson AP, Rigden DJ, Willis AC, Ferguson... in the small subunit (NrfH) of the Wolinella succinogenes cytochrome c nitrite reductase complex FEBS Lett 522, 83– 87 35 Allen JWA & Ferguson SJ (2006) What is the substrate specificity of the System I cytochrome c biogenesis apparatus? Biochem Soc Trans 34, 150–151 36 Bernard DG, Gabilly ST, Dujardin G, Merchant S & Hamel PP (2003) Overlapping specificities of the mitochondrial cytochrome c and c1 heme. .. plasmid and the plasmid for each of the cytochromes, respectively Cells were grown overnight at 37 C with vigorous shaking, in 50 mL of 2· TY medium (16 gÆL)1 peptone, 10 gÆL)1 yeast extract, 5 gÆL)1 NaCl) supplemented with 100 lgÆmL)1 ampicillin, 34 lgÆmL)1 chloramphenicol and 1 mm isopropyl-thio-b-dgalactoside Nine separate cultures were grown for each combination of heme lyase and cytochrome The E coli... Haem staining in gels, a useful tool in the study of bacterial c- type cytochromes Biochim Biophys Acta 852, 288–294 48 Brunger AT (1992) Free R value: a novel statistical ¨ quantity for assessing the accuracy of crystal structures Nature 355, 472–475 49 Cruickshank DW (1999) Remarks about protein structure precision Acta Crystallogr 55, 583–601 50 Read RJ (1986) Improved Fourier coefficients for maps... bloodstream form Trypanosoma theileri Eukaryot Cell 6, 1693–1696 Guerra DG, Decottignies A, Bakker BM & Michels PA (2006) The mitochondrial FAD-dependent glycerol-3phosphate dehydrogenase of Trypanosomatidae and the glycosomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp Mol Biochem Parasitol 149, 155–169 van Hellemond JJ, Simons B, Millenaar FF & Tielens AG (1998) A gene encoding the. .. et al ¨ ¨ St Edmund Hall, Oxford We thank N Brown for very helpful crystallographic discussions, A Holehouse for preliminary experiments, and H Lill for the gift of plasmids encoding the S cerevisiae proteins used in this work Crystallographic data were collected at beamline IO2 at Diamond Light Source, UK, and we acknowledge the support of T Sorensen at Diamond and A Labrador at the ESRF, France Structure . Structure of a trypanosomatid mitochondrial cytochrome c with heme attached via only one thioether bond and implications for the substrate recognition requirements. (histidine and methionine in each case), the cysteines that form thioether bonds to the heme, and the methyl group of the alanine of the C. fasciculata AXXCH heme- binding motif,