The Mitochondrial Cytochrome C N-Terminal Region Is Critical for Maturation by Holocytochrome C Synthase ⇑ Julie M

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

FEBS Letters 585 (2011) 1891–1896

journal homepage: www.FEBSLetters.org

The mitochondrial cytochrome c N-terminal region is critical for maturation by holocytochrome c synthase ⇑ Julie M. Stevens , Yulin Zhang, Gajanthan Muthuvel, Katharine A. Sam, James W.A. Allen, ⇑ Stuart J. Ferguson

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

article info abstract

Article history: The covalent attachment of heme to mitochondrial cytochrome c is catalysed by holocytochrome c Received 24 February 2011 synthase (HCCS, also called heme lyase). How HCCS functions and recognises the substrate apocyto- Revised 18 April 2011 chrome is unknown. Here we have examined HCCS recognition of a chimeric substrate comprising a Accepted 20 April 2011 short mitochondrial cytochrome c N-terminal region with the C-terminal sequence, including the Available online 8 May 2011 CXXCH heme-binding motif, of a bacterial cytochrome c that is not otherwise processed by HCCS. Edited by Peter Brzezinski Heme attachment to the chimera demonstrates the importance of the N-terminal region of the cytochrome. A series of variants of a mitochondrial cytochrome c with amino acid replacements in the N-terminal region have narrowed down the speciﬁcity determinants, providing insight into HCCS Keywords: Holocytochrome c synthase substrate recognition. Heme lyase Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Cytochrome c Substrate recognition

1. Introduction covalently to cytochromes c [5–8], a process that requires catalysis. Holocytochrome c synthase (HCCS; also called heme lyase) is HCCS has not been puriﬁed and its structure and precise molec- the protein in fungi, metazoa and some protists that is responsi- ular function are unknown. A key question regarding the function ble for the covalent attachment of heme to the essential mito- of HCCS is how it recognises the apocytochrome c substrate. HCCS chondrial respiratory protein cytochrome c. Cytochrome c binds is known not to attach heme to bacterial cytochromes [9], includ-

heme covalently at a characteristic CXXCH motif (shown in the ing cytochrome c550 from Paracoccus denitriﬁcans, even though the sequence alignment in Fig. 1) and functions in electron transfer latter has a very similar fold to mitochondrial cytochrome c

between the cytochrome bc1 complex and cytochrome c oxidase [10,11]. Trypanosome cytochrome c, which has a nearly identical in the electron transport chain. The two ligands to the heme iron structure to yeast cytochrome c, is also scarcely recognised by are the H in the CXXCH motif and the M in the C-terminal re- yeast HCCS [12]. A series of variant yeast cytochromes c, in which gion TKM sequence. Initial identiﬁcation of HCCS was in fungal various amino acid sequences were deleted, have provided some species where there is also an isozyme for handling cytochrome insight into which regions of the protein are important for the

c1 (HCC1S) [1,2]. Several studies on HCCS have revealed an combined process of mitochondrial import of apocytochrome c essential role for the protein in mitochondrial import of apocyto- and recognition by HCCS [13]. chrome c [3,4]. HCCS is located in the mitochondrial intermem- Here we have utilised a system in which HCCS functions in the brane space, the compartment where holocytochrome c is Escherichia coli cytoplasm where there are no endogenous c-type produced following import of the apocytochrome from the cyto- cytochromes. An advantage of using a bacterial system to examine sol and transport of the heme cofactor from its site of synthesis processing of cytochrome c variants is that the latter can, in sepa- in the mitochondrial matrix. This seemingly simple post-transla- rate experiments, be targeted to the periplasm for heme attach- tional modiﬁcation system (called System III) contrasts with the ment by the E. coli cytochrome c maturation (Ccm) system, thus complex multi-protein systems (referred to as Systems I, II and providing a route to identifying features important for HCCS activ- IV) that occur in other organisms and organelles to attach heme ity but which are unimportant for either the Ccm system or the folding of the holoprotein. Testing HCCS activity in E. coli also allows separation of the effects of mutations on mitochondrial cytochrome c import (irrelevant in E. coli where the apocytochrome, ⇑ Corresponding authors. Fax: +44 (0) 1865 613201. E-mail addresses: [email protected] (J.M. Stevens), stuart.ferguson heme and HCCS are produced in the same compartment) versus ef- @bioch.ox.ac.uk (S.J. Ferguson). fects on the heme attachment reaction.

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.04.058 1892 J.M. Stevens et al. / FEBS Letters 585 (2011) 1891–1896

Fig. 1. Amino acid sequence alignment of horse cytochrome c, Paracoccus denitriﬁcans cytochrome c550 and Saccharomyces cerevisiae iso-1 cytochrome c. The cysteines of the CXXCH heme-binding motif are highlighted in grey. The amino acids replaced by alanine in this work in the horse and yeast proteins are in bold. The 1 at the top indicates residue 1 of the horse protein (counting after the N-terminal methionine, which is cleaved from the mature protein) and the 1 in bold below the sequences indicates residue 1 of the S. cerevisiae protein. Thus the N-terminal phenylalanine residue is 10 in the horse sequence and 15 in yeast. For mitochondrial cytochrome c in general, G1, G6, F10 and Q16 (horse numbering) are essentially completely conserved. The conserved TKM sequence in which the M is an axial ligand to the heme iron is underlined. GI numbers are indicated next to the sequences.

2. Materials and methods encoded Ccm genes. E. coli strain DH5a was used for routine molecular biology. pKSH028 (encoding the yeast cytochrome c 2.1. Plasmids and strains N-terminal sequence including the CLQCHT heme-binding motif, linked to the P. denitrificans cytochrome c550 C-terminal sequence) Plasmids and strains used in this work are shown in Table 1. Oli- was used as a template for mutagenesis to produce the plasmid gonucleotides were produced by Sigma–Genosys. E. coli strain pYZ01 that encodes the chimera studied in this work, which has BL21(DE3) was used as the host for studies of HCCS activity in the bacterial CKACHM heme-binding region followed by the the cytoplasm both for the single plasmid system (in which the remainder of the bacterial sequence (Fig. 2A). The construction of horse cytochrome c and Saccharomyces cerevisiae HCCS are pKSH028 involved PCR amplifying the first 75 bases of S. cerevisiae encoded on the same plasmid, [14]) and the two-plasmid system cyc1 with primers containing BamHI and XbaI restriction sites. The (in which the cytochrome c and S. cerevisiae HCCS are on separate DNA encoding P. denitrificans cytochrome c550, excluding the N- plasmids [9]). Periplasmic holocytochrome formation was exam- terminus up to and including the CXXCH motif, was amplified with ined in the E. coli strain EC06 [15], which has a chromosomal dele- primers containing XbaI and HindIII sites. These two fragments tion of the ccm operon, in the presence or absence of plasmid- were subcloned into pTZ19R (Fermentas) and the fused DNA was inserted into pET3a (Novagen) using NdeI and BamHI restriction sites, producing pKSH028. KOD polymerase (Novagen) was used for Quikchange site-directed mutagenesis and cloning. DNA Table 1 constructs were sequenced before use (Geneservice, Oxford). Plasmids and strains. pCYCSperiWT was produced by gene synthesis (Genscript) Name Description Source and contains the PelB periplasmic targeting sequence EC06 E. coli strain DccmABCDEFGH [15] (KYLLPTAAAGLLLLAAQPAMA) on the N-terminus of horse cyto- pEC86 E. coli ccmABCDEFGH,CmR [19] chrome c in the vector pKK223–3. pWT S. cerevisiae cyc3, Equus caballus CYCS, AmpR [14] cytochrome c pOScyc1 S. cerevisiae cyc1, AmpR [9] 2.2. Bacterial cultures and protein extraction pACcyc3 S. cerevisiae cyc3, CmR [9] pKSH028 S. cerevisiae cyc1 N-terminus up to This work Routine cell culturing was performed in Luria–Bertani medium CXXCHX + P. denitrificans c550 C-terminus, supplemented with antibiotics where required (ampicillin at AmpR 100 lgmlÀ1 and chloramphenicol at 34 lgmlÀ1). Cultures were pYZ01 S. cerevisiae cyc1 N-terminus + P. denitrificans This work R grown at 37 °C with shaking at 200 rpm for 20 h before harvesting. c550 C-terminus from CXXCH, Amp pGM01 pWT cytochrome c with D2A, AmpR This work For analysis of the horse and yeast cytochrome c variants, 20 ml pGM02 pWT cytochrome c with E4A, AmpR This work cultures were grown at 30 °C for 20 h. For preparation of periplas- R pYZ07 pWT cytochrome c with K5A, Amp This work mic fractions, a published method was used [16]. BugBuster (Nova- pGM03 pWT cytochrome c with G6A, AmpR This work pGM04 pWT cytochrome c with K7A, AmpR This work gen) was used to lyse the sphaeroplasts produced after the removal pYZ08 pWT cytochrome c with K8A, AmpR This work of the periplasmic fraction. 1 mM isopropyl-1-thio-b-D-galactopy- pGM05 pWT cytochrome c with F10A, AmpR This work ranoside (IPTG) was used to induce expression of the periplasmi- R pKK223–3 Cloning vector, Amp Pharmacia cally targeted proteins. IPTG induction did not significantly pCYCSperiWT E. caballus CYCS in pKK223–3 with periplasmic This work enhance the production of holocytochrome in the cytoplasmic con- targeting sequence, AmpR pCYCSperiD2A pCYCSperiWT with D2A, AmpR This work structs and thus was not used. At least five replicate cultures were pCYCSperiE4A pCYCSperiWT with E4A, AmpR This work analysed for each experiment. pCYCSperiG6A pCYCSperiWT with G6A, AmpR This work R pCYCSperiK7A pCYCSperiWT with K7A, Amp This work 2.3. Analysis of cytochrome content pCYCSperiF10A pCYCSperiWT with F10A, AmpR This work pYZ02 pOScyc1 with G6A, AmpR This work pYZ03 pOScyc1 with K10A, AmpR This work Extracts were analysed by SDS–PAGE (pre-cast 10% Bis–Tris pYZ04 pOScyc1 with G11A, AmpR This work gels, Invitrogen) followed by heme staining [17], a method which R pYZ05 pOScyc1 with F15A, Amp This work detects the presence of covalently bound heme. UV–vis spectros- pYZ06 pOScyc1 with R18A, AmpR This work copy was performed on a Perkin–Elmer Lambda 2 UV–vis spectro- J.M. Stevens et al. / FEBS Letters 585 (2011) 1891–1896 1893

A TEFKAGSAKKGATLFKTRRCCKACHMIQAPDGTDIIKGGKTGPNLYGVVKACHMIQAPDGTDIIKGGKTGPNLYGVV GRKIASEEGFKYGEGILEVAEKNPDLTWTEADLIEYVTDPKPWLVKMGRKIASEEGFKYGEGILEVAEKNPDLTWTEADLIEYVTDPKPWLVKM TTDDKGAKTKMTFKMGKNQADVVAFLAQNSPDAGGDGEAAAEGESNDDKGAKTKMTFKMGKNQADVVAFLAQNSPDAGGDGEAAAEGESN

B C M 1 2

Reduced protein Pyridine hemochrome absorbance

wavelength (nm)

Fig. 2. (A) The amino acid sequence of the chimeric cytochrome c. The sequence in blue is the S. cerevisiae iso-1 cytochrome c N-terminal sequence and that highlighted in grey is the sequence of the cytochrome c550 from P. denitrificans (excluding the amino acids N-terminal to CKACH). (B) The yeast-bacterial cytochrome c chimera (shown in A) analysed by SDS–PAGE stained for covalently attached heme. M shows molecular mass markers (38, 28, 14, 6 kDa from the top). Lane 1 shows the purified chimeric protein; lane 2 shows pure yeast iso-1 cytochrome c (mass = 12 588 Da, Sigma) for comparison. (C) Spectroscopic analysis of the purified chimeric yeast-bacterial cytochrome. The spectrum on reduction with disodium dithionite is shown in blue and that following the addition of 0.15 M NaOH and 19% (vol/vol) pyridine shown in red. In the spectrum of the reduced protein the wavelength maxima are 415, 520 and 550 nm. The a-band is at 550 nm in the pyridine hemochrome spectrum.

photometer. Cell extracts or pure protein samples were reduced by of the protein could be key to substrate recognition. As a ﬁrst step the addition of a few grains of disodium dithionite (Sigma) for to establishing the role of this sequence experimentally we have spectroscopic analysis. Data for the cytochrome c variants were constructed a chimeric protein comprising the N-terminal 18 ami- normalised according to the wet weight of the cultured cells. no acids of iso-1 cytochrome c (CYC1) from S. cerevisiae followed by

the C-terminal sequence of cytochrome c550 from P. denitrificans 2.4. Purification of chimeric protein (the CXXCH heme-binding motif and residues C-terminal to it), as shown in Fig. 2A. The chimera was coexpressed in the E. coli The cytoplasmic fraction (from 2 l of E. coli BL21(DE3) induced cytoplasm with S. cerevisiae HCCS from a separate plasmid (pAC- at OD600 0.8 with 1 mM IPTG) was obtained using BugBuster cyc3). The periplasmic fraction was removed from the harvested (Novagen) according to the manufacturer’s instructions. The cyto- cells to avoid interference by endogenous cytochromes in that plasm was applied to an 80 ml DEAE-Sepharose Fast-Flow (GE compartment. The cytoplasmic extract was red and was fraction- Healthcare) column equilibrated with 50 mM Tris–HCl, pH 8 and ated by anion-exchange chromatography. The pooled red fractions washed with the same buffer. The chimeric cytochrome was eluted were analysed by electrospray mass spectrometry, which showed with a 300 ml linear salt gradient (0–500 mM NaCl in 50 mM Tris– the predominant protein species to have a mass of 15435.6 Da HCl, pH 8) at a flow rate of 2 ml/min, collecting 5 ml fractions. Frac- (±3.1 Da). The predicted mass of the chimeric protein, (which has tions containing the holocytochrome were pooled and concen- its N-terminal methionine cleaved, as would be expected in the trated; the overall yield of protein was 10 mg from 2 l of culture. E. coli cytoplasm) is 14,816 + 616.5 Da for heme, resulting in a total mass of 15,432.5 Da, within experimental error of the value ob- 3. Results and discussion served. The protein was analysed by UV–vis spectroscopy and SDS–PAGE followed by heme-staining (Fig. 2B and C). These results 3.1. Chimera of yeast cytochrome c N-terminal region and bacterial demonstrate that S. cerevisiae HCCS is able to attach heme to the cytochrome c550 C-terminal region chimeric protein as shown both by the heme-staining band on SDS–PAGE in the expected position and the mass spectrometry Previous substrate specificity studies of S. cerevisiae HCCS have experiment. The position of the a-band in the pyridine hemo- shown that the enzyme does not attach heme to cytochrome c550 chrome spectrum at 550 nm indicates that the two vinyl groups from P. denitrificans [9]. The three-dimensional structures of cyto- of the heme are saturated, consistent with attachment to the heme chrome c550 and mitochondrial cytochromes c are very similar via two thioether bonds, as observed in typical c-type cytochromes [10,11], eliminating recognition of the overall protein fold as the [1]. It is notable that the chimeric protein has folded into a stable, major criterion for substrate recognition by HCCS. The N-terminal soluble conformation. region (which in this work refers to the sequence on the N-termi- The N-terminal region of cytochrome c is therefore critical for nal side of the CXXCH heme-binding motif) is a highly conserved processing by HCCS since replacement by a yeast sequence of the part of mitochondrial cytochromes c (some examples are shown residues to the N-terminal side of the CXXCH motif of a bacterial in Fig. 1 and its legend). This conservation, along with the close protein, which S. cerevisiae HCCS cannot process, allows significant proximity to the heme-binding motif, indicates that this region expression of the chimeric cytochrome c. We note that the ‘‘XX’’ 1894 J.M. Stevens et al. / FEBS Letters 585 (2011) 1891–1896 residues, between the heme-binding cysteines (Fig. 1) of the and K7A variants, heme attachment to these proteins was not mitochondrial protein, are relatively highly conserved. AQ is very attenuated by the amino acid replacements (in fact, the levels of common in mammalian cytochromes c and S. cerevisiae iso-1- holocytochrome were higher than the wild-type in the latter two cytochrome c is LQ, but the occurrence of the very different KA cases). We note that the apparent increase in the level of holocyto- in the chimeric protein does not present a significant barrier to chrome produced for E4A and K7A could be due to the horse cyto- the heme attachment process. The QCH sequence is present in chrome c sequence having been made more, in terms of charge, almost all mitochondrial cytochromes c but the Q is clearly not like yeast cytochrome c (the natural substrate of the HCCS used). essential for processing by HCCS. E4 in the horse sequence aligns with a lysine in the yeast sequence and K7 with alanine in yeast (Fig. 1). In contrast, the G6A and F10A 3.2. Substitution of conserved amino acids in the N-terminal region of variants were not matured at levels detectable by heme-staining mitochondrial cytochrome c (which detects as little as 1 pmol of heme [18]). K5A and K8A variants of horse cytochrome c were also matured at similar levels to The results above show that the N-terminal 18 amino acids of the wild type protein (data not shown). S. cerevisiae mitochondrial cytochrome c contain a specificity In order to demonstrate that the absence of holocytochrome determinant for S. cerevisiae HCCS. In order to narrow down the with the G6A and F10A variants was a consequence of lack of pro- specificity elements in the N-terminal cytochrome c sequence, cessing by HCCS, and not due to intrinsic protein instability, plas- seven mutations were made in the horse cytochrome c gene in a mids were constructed that expressed these and other variants of single plasmid system from which the cytochrome is coexpressed horse cytochrome c with periplasmic targeting sequences. These with yeast HCCS [14]. These were D2A, E4A, K5A, G6A, K7A, K8A were tested by coexpression with the E. coli cytochrome c matura- and F10A (indicated in Fig. 1). These residues are among the most tion genes (from the plasmid pEC86 [19]) for which there is strong highly conserved in this region of mitochondrial cytochromes c and evidence that little more than the CXXCH motif of the apocyto- all but D2 are in the N-terminal a-helix in the folded holocytochrome is recognised [20,21]. SDS–PAGE analysis and heme-stain- chrome. The single plasmid system was selected because it has ing of extracts of cells expressing the wild-type and five variant been optimised for high levels of horse holocytochrome c produc- cytochromes c is shown in Fig. 3B. The larger size of the proteins tion [14] and the horse cytochrome c has a shorter and therefore observed on the gel relative to the pure horse cytochrome c indi- more tractable N-terminal region than the yeast protein. High lev- cates that cleavage of the PelB periplasmic targeting sequence els of cytochrome c production in this system would enable any has not occurred efficiently; the signal peptide adds 2500 Da to perturbations or partial attenuations of holocytochrome c produc- the mass of the proteins. Incomplete cleavage of the PelB sequence tion to be identified. The extent of sequence similarity in the has been reported [22]. E. coli EC06 lacking either pEC86 or a plas- conserved C-terminal regions of the horse and S. cerevisiae HCCS mid encoding a cytochrome c did not show any heme staining is 61%, which is consistent with the ability of the yeast HCCS to bands equivalent to those from mitochondrial cytochrome c. The process horse cytochrome c. variant cytochromes are processed at comparable levels to the Analysis of cytoplasmic extracts of E. coli expressing the variant wild-type, indicating that the G6A and F10A variants are produced cytochromes by SDS–PAGE and heme-staining is shown in Fig. 3A. and capable of undergoing heme attachment. These two amino Duplicate extracts for each protein are shown. For the D2A, E4A acids are therefore important for the interaction between the

Fig. 3. (A) SDS–PAGE analysis followed by heme staining of cytoplasmic extracts of E. coli coexpressing HCCS with wild-type horse cytochrome c and N-terminal region variants. Lanes labelled M show molecular mass markers (28, 14 and 6 kDa from the top). Lanes 1 and 8 show wild-type cytochrome c, lanes 2 and 3 show the D2A variant, lanes 4 and 5 the E4A variant, lanes 6 and 7 the G6A variant, lanes 9 and 10 the K7A variant, lanes 11 and 12 the F10A variant. The expected molecular mass of horse cytochrome c produced in this work is 12,303 Da (including the heme). Equal amounts of total protein were loaded into each lane. (B) Analysis of heme attachment to wild- type horse cytochrome c and N-terminal region variants by the E. coli Ccm system (from the plasmid pEC86) in the periplasm of E. coli. Lanes 1 and 7 show periplasmic extracts of cells expressing wild-type cytochrome c, lane 2 is the D2A variant, lane 3 is the E4A variant, lane 4 is the G6A variant, lane 5 is the K7A variant, lane 6 is the F10A variant and lane 8 contains pure native horse cytochrome c (Sigma). The marker proteins are 62, 49, 38, 28, 14 and 6 kDa from the top of the gel (M). The samples loaded were normalised according to the masses of the wet cell pellets. J.M. Stevens et al. / FEBS Letters 585 (2011) 1891–1896 1895 apocytochrome and HCCS, and not essential for a folded holocyto- of cytochrome c in the R18A variant (lane 6) is notable as the res- chrome c. This conclusion would be invalid if mitochondrial cyto- idue next to the first C of the CXXCH heme-binding motif is almost chrome c were able to fold differently in the periplasm than in always positively charged in mitochondrial cytochromes c. HCCS the cytoplasm of E. coli. In such a circumstance, F10 might be appears to be indifferent to the presence of this positive charge. essential for a folding pathway in the cytoplasm of E. coli but not The other two replacements, G6A corresponding to horse G2, and in the periplasm. However, we note that horse mitochondrial cyto- K10A corresponding to horse K5, did not abolish the maturation chrome c folds to the same final structure in the periplasm [23] or of the cytochrome by the HCCS. the cytoplasm [14] of E .coli as in mitochondria. Thus our control It has been shown previously [13] that F15 is important for im- experiment would only be invalidated if different folding pathways port and/or processing of mitochondrial cytochrome c in S. cerevi- were used in different environments to reach the same final struc- siae. Our result indicates that a disruption of apocytochrome-HCCS ture. This is extremely unlikely, not least because from the various interaction in the absence of this side chain could explain the data discussed in the present paper the HCCS system parallels the observation. We note that the side-chain of F15 (yeast)/F10 (horse) Ccm system by catalysing heme attachment to relatively unfolded is relatively buried in the folded protein structure [27] but may be peptides before acquisition of the final 3D fold occurs. It is notable exposed before heme attachment. The conclusions of the present that Rumbley et al. reported that when K8G and V11G variations work are consistent with the findings of Veloso et al. [28],who were made to horse cytochrome c, their expression system, involv- showed that heme was covalently attached by yeast mitochondria ing the yeast HCCS, produced misfolded forms of cytochrome c to a peptide corresponding to the 26 N-terminal residues, includ- with distinct spectra [14]. The data presented do not allow one ing the CXXCH motif, of horse cytochrome c. However, in that work to distinguish between misattachment of heme by HCCS, or mis- it was not shown that proper heme attachment had been achieved. folding following attachment of heme owing to the introduced G, Nye and Scarpulla presented evidence of heme attachment to a fu- a residue that is known to cause perturbations in protein folds. sion of yeast cytochrome c, at its C-terminus, to chloramphenicol Nevertheless their data do show that aberrant mitochondrial holo- acetyl transferase. Having acquired a globular fold the latter was cytochomes c can be detected in experiments similar to ours and retained on the cytosolic side of the outer mitochondrial mem- thus strengthen the case that the absence of any detectable G6A brane with at least part of the cytochrome in the intermembrane and F10A variants in our work is a consequence of failure by HCCS space [29]; the cytochrome thus spanned the outer membrane to process the apoproteins. All the horse mitochondrial cyto- with the N-terminus accessible to the HCCS. This observation sup- chromes c produced in the current work by HCCS functioning in ports the evidence discussed in this paper that it is the N-terminal E. coli had normal UV–vis spectra (except of course the G6A and region that is critical for recognition by HCCS. It is also notable that F10A variants). Others have shown that K8C, K8E and V11C vari- HCCS effectively processes variants of yeast cytochrome c in which ants of horse cytochrome c are matured by yeast HCCS [24,25]. the heme–iron axial methionine ligand is replaced by several res- Despite the high similarity between S. cerevisiae and horse idues, showing that HCCS activity does not HCCS activity does HCCS, and the proven ability of the former to efficiently catalyse not require this C-terminal residue [30]. Previously, we have spec- production of horse cytochrome c [14], single residue variants of ulated that holocytochrome c synthase might act as a type of chap- five conserved N-terminal residues G6A, K10A, G11A, F15A and erone, stabilising the cytochrome’s overall structure to optimise R18A of S. cerevisiae iso-1 cytochrome c were also tested for matu- the proximity of heme vinyl groups to the critical cysteines [31]. ration by S. cerevisiae HCCS. The HCCS and cytochrome proteins The new data presented here argues against this interpretation were expressed from separate plasmids in the E. coli cytoplasm. and supports a model in which HCCS interacts with the N-terminus A heme-stained SDS–PAGE of the cytoplasmic extracts of cells of unfolded apocytochrome and that acquisition of the final fold expressing these variants is shown in Fig. 4. The F15A replacement follows heme attachment. The extent of unfolding of the apocyto- (corresponding to F10 in the horse cytochrome c, shown in Fig. 1) chrome is unclear because previous work has shown that even again caused complete loss of cytochrome c production, as seen in mitochondrial apocytochrome c acquires sufficient tertiary struc- lane 5, compared with the WT in lane 1. However, the yeast G11A ture to bind heme non-covalently [32]. variant (lane 4, corresponding to horse G6A) was matured, as other Despite the importance of mitochondrial cytochrome c in respi- G11 variants are in yeast [26], implying that the context of this G is ration and apoptosis, knowledge of how it is formed by a post- important for its interaction with HCCS. The continued production translational modification reaction catalysed by holocytochrome c synthase is lacking. Demonstration in this work that the yeast-bacterial chimeric cytochrome c is recognised by HCCS, taken together with the inhibitory effect of varying the conserved phenylalanine in the N-terminal region, allows us to conclude that the major, and possibly only, recognition features between the apocytochrome and the synthase reside in the cytochrome N-terminal region, a conclusion that is in good agreement with a variety of ear- lier data in the literature [28,29]. This is not to say that recognition depends only on the phenylalanine. The finding that a glycine is critical for heme attachment to the horse, but not the yeast, protein indicates the overall organisation of the N-terminal region is important, as does the failure of the phenylalanine in the P. denitrif-

icans c550 N-terminal region, at a different spacing relative to the CXXCH motif, to ensure heme attachment by HCCS.

Fig. 4. SDS–PAGE analysis followed by heme staining of cytoplasmic extracts of During revision of the present work, Kleingardner and Bren [33] E. coli coexpressing S. cerevisiae HCCS with wild-type S. cerevisiae cytochrome c and also reported that heme is not attached to the F10A variant of N-terminal region variants. Lanes labelled M show molecular mass markers (14, 28 horse cytochrome c by S. cerevisiae HCCS whereas the holocyto- and 38 kDa from the bottom). Lane 1 shows wild-type cytochrome c, lane 2 the G6A chrome is produced by the Ccm system in the E. coli periplasm in variant, lane 3 the K10Avariant, lane 4 the G11A variant, lane 5 the F15A variant and a state indistinguishable, on the basis of two spectroscopic criteria, lane 6 shows the R18A variant. Equal amounts of total protein were loaded into each lane and were normalised according to wet cell mass. Note that the from the native protein. They further showed that adding an extra recombinant S. cerevisiae cytochrome c runs larger than the commercial protein. glycine residue at position 13 of the horse protein led to misformed 1896 J.M. Stevens et al. / FEBS Letters 585 (2011) 1891–1896 products when HCCS was used but a normal holocytochrome [15] Throne-Holst, M., Thöny-Meyer, L. and Hederstedt, L. (1997) Escherichia coli formed when the protein was processed by the Ccm system. ccm in-frame deletion mutants can produce periplasmic cytochrome b but not cytochrome c. FEBS Lett. 410, 351–355.

[16] Allen, J.W., Barker, P.D. and Ferguson, S.J. (2003) A cytochrome b562 variant Acknowledgements with a c-type cytochrome CXXCH heme-binding motif as a probe of the Escherichia coli cytochrome c maturation system. J. Biol. Chem. 278, 52075– 52083. This work was supported by the Biotechnology and Biological [17] Goodhew, C.F., Brown, K.R. and Pettigrew, G.W. (1986) Haem staining in gels, a Sciences Research Council (Grants BB/H017887/1 and BB/ useful tool in the study of bacterial c-type cytochromes. Biochim. Biophys. D019753/1). G.M. was supported by the SCORE program of UW- Acta 852, 288–294. [18] Goddard, A.D., Stevens, J.M., Rao, F., Mavridou, D.A., Chan, W., Richardson, D.J., Madison. K.A.S. was a WR Miller junior research fellow at St. Ed- Allen, J.W. and Ferguson, S.J. (2010) C-Type cytochrome biogenesis can occur mund Hall, Oxford. J.W.A.A. is a BBSRC David Phillips fellow. We via a natural Ccm system lacking CcmH, CcmG, and the heme-binding thank Dr. David Staunton for mass spectrometry. Plasmids were histidine of CcmE. J. Biol. Chem. 285, 22882–22889. [19] Arslan, E., Schulz, H., Zufferey, R., Kunzler, P. and Thony-Meyer, L. (1998) kindly provided by Prof. W. Englander (pWT cytochrome c [14]), Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits

Prof. H. Lill (pOScyc1 and pACcyc3 [9]) and Prof. L. Thöny-Meyer of the cbb3 oxidase in Escherichia coli. Biochem. Biophys. Res. Commun. 251, (pEC86 [19]). 744–747. [20] Braun, M. and Thony-Meyer, L. (2004) Biosynthesis of artificial microperoxidases by exploiting the secretion and cytochrome c maturation References apparatuses of Escherichia coli. Proc. Natl. Acad. Sci. USA 101, 12830–12835. [21] Ferguson, S.J., Stevens, J.M., Allen, J.W. and Robertson, I.B. (2008) Cytochrome c [1] Dumont, M.E., Ernst, J.F., Hampsey, D.M. and Sherman, F. (1987) Identification assembly: a tale of ever increasing variation and mystery? Biochim. Biophys. and sequence of the gene encoding cytochrome c heme lyase in the yeast Acta 1777, 980–984. Saccharomyces cerevisiae. EMBO J. 6, 235–241. [22] Mavridou, D.A., Stevens, J.M., Goddard, A.D., Willis, A.C., Ferguson, S.J. and [2] Drygas, M.E., Lambowitz, A.M. and Nargang, F.E. (1989) Cloning and analysis of Redfield, C. (2009) Control of periplasmic interdomain thiol:disulfide the Neurospora crassa gene for cytochrome c heme lyase. J. Biol. Chem. 264, exchange in the transmembrane oxidoreductase DsbD. J. Biol. Chem. 284, 17897–17906. 3219–3226. [3] Nicholson, D.W., Kohler, H. and Neupert, W. (1987) Import of cytochrome c [23] Kellogg, J.A. and Bren, K.L. (2002) Characterization of recombinant horse into mitochondria. Cytochrome c heme lyase. Eur. J. Biochem. 164, 147–157. cytochrome c synthesized with the assistance of Escherichia coli cytochrome c [4] Nargang, F.E., Drygas, M.E., Kwong, P.L., Nicholson, D.W. and Neupert, W. maturation factors. Biochim. Biophys. Acta 1601, 215–221. (1988) A mutant of Neurospora crassa deficient in cytochrome c heme lyase [24] Pepelina, T.Y., Chertkova, R.V., Ostroverkhova, T.V., Dolgikh, D.A., Kirpichnikov, activity cannot import cytochrome c into mitochondria. J. Biol. Chem. 263, M.P., Grivennikova, V.G. and Vinogradov, A.D. (2009) Site-directed 9388–9394. mutagenesis of cytochrome c: reactions with respiratory chain components [5] Sanders, C., Turkarslan, S., Lee, D.W. and Daldal, F. (2010) Cytochrome c and superoxide radical. Biochemistry (Mosc) 74, 625–632. biogenesis: the Ccm system. Trends Microbiol. 18, 266–274. [25] Tenger, K., Khoroshyy, P., Kovacs, K.L., Zimanyi, L. and Rakhely, G. (2007) [6] Kranz, R.G., Richard-Fogal, C., Taylor, J.S. and Frawley, E.R. (2009) Cytochrome c Improved system for heterologous expression of cytochrome c mutants in biogenesis: mechanisms for covalent modifications and trafficking of heme Escherichia coli. Acta Biol. Hung. 58 (Suppl.), 23–35. and for heme-iron redox control. Microbiol. Mol. Biol. Rev. 73, 510–528. [26] Hampsey, D.M., Das, G. and Sherman, F. (1988) Yeast iso-1-cytochrome c: [7] Giege, P., Grienenberger, J.M. and Bonnard, G. (2008) Cytochrome c biogenesis genetic analysis of structural requirements. FEBS Lett. 231, 275–283. in mitochondria. Mitochondrion 8, 61–73. [27] Banci, L., Bertini, I., Huber, J.G., Spyroulias, G.A. and Turano, P. (1999) Solution [8] Allen, J.W., Jackson, A.P., Rigden, D.J., Willis, A.C., Ferguson, S.J. and Ginger, M.L. structure of reduced horse heart cytochrome c. J. Biol. Inorg. Chem. 4, 21–31. (2008) Order within a mosaic distribution of mitochondrial c-type cytochrome [28] Veloso, D., Juillerat, M. and Taniuchi, H. (1984) Synthesis of a heme fragment biogenesis systems? FEBS J. 275, 2385–2402. of horse cytochrome c which forms a productive complex with a native [9] Sanders, C. and Lill, H. (2000) Expression of prokaryotic and eukaryotic apofragment. J. Biol. Chem. 259, 6067–6073. cytochromes c in Escherichia coli. Biochim. Biophys. Acta 1459, 131–138. [29] Nye, S.H. and Scarpulla, R.C. (1990) Mitochondrial targeting of yeast apo iso-1- [10] Timkovich, R. and Dickerson, R.E. (1976) The structure of Paracoccus cytochrome c is mediated through functionally independent structural

denitrificans cytochrome c550. J. Biol. Chem. 251, 4033–4046. domains. Mol. Cell. Biol. 10, 5763–5771. [11] Tanaka, N., Yamane, T., Tsukihara, T., Ashida, T. and Kakudo, M. (1975) The [30] Silkstone, G., Stanway, G., Brzezinski, P. and Wilson, M.T. (2002) Production crystal structure of bonito (katsuo) ferrocytochrome c at 2.3 Å resolution. II. and characterisation of Met80X mutants of yeast iso-1-cytochrome c: spectral, Structure and function. J. Biochem. 77, 147–162. photochemical and binding studies on the ferrous derivatives. Biophys. Chem. [12] Fulop, V., Sam, K.A., Ferguson, S.J., Ginger, M.L. and Allen, J.W. (2009) Structure 98, 65–77. of a trypanosomatid mitochondrial cytochrome c with heme attached via only [31] Daltrop, O., Allen, J.W., Willis, A.C. and Ferguson, S.J. (2002) In vitro formation one thioether bond and implications for the substrate recognition of a c-type cytochrome. Proc. Natl. Acad. Sci. USA 99, 7872–7876. requirements of heme lyase. FEBS J. 276, 2822–2832. [32] Daltrop, O. and Ferguson, S.J. (2003) Cytochrome c maturation. The in vitro [13] Wang, X., Dumont, M.E. and Sherman, F. (1996) Sequence requirements for reactions of horse heart apocytochrome c and Paracoccus dentrificans mitochondrial import of yeast cytochrome c. J. Biol. Chem. 271, 6594–6604. apocytochrome c550 with heme. J. Biol. Chem. 278, 4404–4409. [14] Rumbley, J.N., Hoang, L. and Englander, S.W. (2002) Recombinant equine [33] Kleingardner, J.G. and Bren, K.L. (2011) Comparing substrate specificity cytochrome c in Escherichia coli: high-level expression, characterization, and between cytochrome c maturation and cytochrome c heme lyase systems folding and assembly mutants. Biochemistry 41, 13894–13901. for cytochrome c biogenesis. Metallomics 3, 396–403.