|Home | About | Journals | Submit | Contact Us | Français|
The primary sequence of cytochrome c 6 from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 Å resolution. The crystal belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 84.58, c = 232.91 Å and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c 6 were most similar to those of red algal cytochrome c 6. These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.
Soluble c-type monohaem cytochromes are ubiquitously distributed haem proteins which act as electron carriers in mitochondria, bacteria, algal chloroplasts and cyanobacteria. Cytochrome c 6 is a soluble low-spin haem protein that functions in oxygenic photosynthesis as an electron carrier between cytochrome f, which is part of the membrane-embedded cytochrome b 6 f complex, and the P700 reaction centre of photosystem I (Kerfeld & Krogmann, 1998 ). This cytochrome c 6 is classified as a class I c-type cytochrome, in which the haem iron has histidine–methionine axial coordination. Plastocyanin is a blue copper protein with the same function as cytochrome c 6. Cytochrome c 6 and plastocyanin have completely different amino-acid sequences and secondary and tertiary structures, but they contain similar acidic and hydrophobic patches on their surface for recognition of their interaction partners (Frazão et al., 1995 ; Ullmann et al., 1997 ).
Although chloroplasts are thought to have evolutionally arisen from cyanobacteria (Aitken, 1976 ), there are differences in the expression and genome coding of cytochrome c 6 in green and red algae. In the green alga Chlamydomonas reinhardtii, the gene for cytochrome c 6 exists in the genomic DNA and its coding region is interrupted by two introns (Hill et al., 1991 ). On the other hand, in the red alga Porphyra purpurea the petJ gene encoding cytochrome c 6 exists in the chloroplast genome (Reith & Munholland, 1993 ).
In eukaryotic brown algae, which contain no plastocyanin, photosynthetic electron transport between cytochrome f and photosystem I is only performed by cytochrome c 6. It is generally considered that brown algae acquired their chloroplasts via secondary endosymbiosis involving a primitive red algal endosymbiont and a nonphotosynthetic eukaryote host (Cavalier-Smith, 2000 ; McFadden, 1999 ). Although the physicochemical properties and amino-acid sequences of cytochromes c 6 from the brown algae Petalonia fascia and Alaria esculenta have been determined (Sugimura et al., 1981 ; Laycock, 1975 ), the genome code and tertiary structure of brown algal cytochrome c 6 remain to be studied. In this study, we determined the genome code of the brown algal cytochrome c 6 gene from the brown alga Hizikia fusiformis, determined the crystal structure of the protein and compared it with those of cyanobacterial and red and green algal cytochromes c 6.
The brown alga H. fusiformis was collected in the coastal area off Hayama, Japan. Total RNA was isolated from the brown alga using the RNeasy Plant Mini Kit (Qiagen). Poly(A)+ mRNA and poly(A)− mRNA were separated from the total RNA using Oligotex-dT30 (Takara). Additional of adenine at the 3′-terminus of poly(A)− mRNA was carried out for 40 min at 310 K in a reaction mixture containing 2 µg poly(A)− mRNA, 50 mM Tris–HCl pH 7.9, 50 mM MgCl2, 10 mM MnCl2, 500 mM NaCl, 2.5 mM DTT, 0.5% BSA, 1 mM ATP, 121 U ribonuclease inhibitor and 1.5 U poly(A) polymerase (Takara). First-strand cDNA was synthesized using a 1st Strand cDNA Synthesis Kit with AMV Reverse Transcriptase (Life Science Inc.) and the oligonucleotide primer 5′-CGGGATCC(T)25-3′, designated primer P1 (reverse). To obtain the clone encoding the 3′-region of cytochrome c 6 from H. fusiformis, we designed the degenerate oligonucleotide primer P2 (forward), 5′-AAYTGYGCIGCIGCITGYCAYGCI-3′, based on the highly conserved residues around the haem c motif (Asn-Cys-Ala-Ala-Cys-His-Ala) of cytochrome c 6 from the cyanobacteria Synechocystis PCC6803 and Anabaena 7119, the green alga C. reinharditii, the euglena Euglena gracillis and the cyanelle Cyanophora paradoxa. PCR products were subcloned into a pGEM T-Easy vector (Promega). DNA sequencing was performed by the dideoxy chain-termination method using a Thermo Sequence fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amasham) and an automated DSQ 2000L DNA sequencer (Shimadzu, Japan). The first-strand cDNA from H. fusiformis were dC-tailed at their 3′-ends using the 5′ RACE system for Rapid Amplification of cDNA Ends Reagent Assembly v.2.0 (Life Technologies Inc.). The 5′-region of the cytochrome c 6 gene from H. fusiformis was amplified by the polymerase chain reaction (PCR) using a forward primer complementary to the dC tail [P3, 5′-GGCCACGCGTCGACTAGTAC(G)16-3′] and a gene-specific primer designed based on the 3′-region sequences of the cytochrome c 6 cDNA (Hf1, 5′-TCAGGCATAATAATAACATTATTACCGCC-3′). The PCR product was subcloned and sequenced by the same methods as used in 3′ RACE. Genome DNA from H. fusiformis was extracted using Isoplant II (Nippon Gene). To obtain the genome sequence of H. fusiformis cytochrome c 6, we designed gene-specific primers for amplification of the full-length cytochrome c 6 gene on the basis of the cDNA sequence of cytochrome c 6 (Hf2, 5′-ATGGGGGGGTGGAAAATTTATTATT-3′, forward; Hf3, 5′-TCAACGTTCCAGGTCCAATAATATCATAA-3′, reverse). The PCR product was subcloned and sequenced according to 3′ RACE.
Construction and overproduction of the cytochrome c 6 gene (petJ) in Escherichia coli was performed according to the method described by Satoh et al. (2002 ) with slight modifications. The mature cytochrome c 6 sequence was amplified using the forward primer ExP1 (5′-CATGCCATGGGCTGATATTAATCATGGAG-3′) corresponding to codons for the amino-acid residues of the cytochrome c 6 N-terminal region and the reverse primer ExP2 (5′-GCGGATCCTTAGTTCCAACTCTTTTCAG-3′) corresponding to codons for the amino-acid residues of the C-terminal region. The amplified mature cytochrome c 6 sequence was ligated to the pelB signal sequence adapter (Genset Co. Ltd). The resulting pelB–cytochrome c 6 hybrid gene was cloned into NdeI–BamHI sites of pET22b(+) (Novagen Co. Ltd) to construct the plasmid pET22bHfc6.
The cytochrome c maturation genes ccmA–H were amplified using the polymerase chain reaction from E. coli MC1061 genomic DNA using the forward primer P3, 5′-CCAGAATTCGGTTGCCGCGAAGATGCAT-3′, corresponding to upstream of the ccmA gene from the E. coli K12 MG1655 genome sequence (AE000309), and the reverse primer P4, 5′-TTCCTGCAGCAACGCGGGGCACAATAAA-3′, corresponding to downstream of the ccmH gene. The resulting ccmA–H gene was cloned into the EcoRI–PstI sites of pSTV28 (Takara Shuzo Co.) to create the plasmid pSTV28ccmA–H.
For the overproduction of H. fusiformis cytochrome c 6, both pET22bHfc6 and pSTV28ccmA–H were co-introduced into E. coli BL21 (DE3). Transformed E. coli cells were grown in 1 l Luria–Bertani (LB) medium supplemented with 100 mg l−1 ampicillin and 20 mg l−1 chloramphenicol at 303 K for 36 h. Cells were harvested by centrifugation at 6000g (277 K) for 5 min. The pellet was resuspended in 80 ml PBS buffer and disrupted using a high-pressure homogenizer (Mini Lab 8.30H, Rannie). The suspension was fractionated with ammonium sulfate (40–80% saturation). The precipitate was dissolved in a small amount of 20 mM sodium acetate buffer pH 5.5 and dialyzed against the same buffer. The sample was applied onto a DE52 cellulose column (Whatman, 2.0 × 40.0 cm) equilibrated with 20 mM sodium acetate buffer pH 5.5. After the column had been washed with the same buffer, the proteins were eluted using a linear gradient of sodium acetate pH 5.5 (20–200 mM). Fractions containing cytochrome c 6 were pooled and dialyzed against 20 mM sodium acetate buffer pH 5.5 and the dialyzed sample was applied onto a Poros HQ/20 column (Applied Biosystems) previously equilibrated with the same buffer. After the column had been washed with the same buffer, the proteins were eluted with an NaCl gradient (0–500 mM) in the same buffer. The sample thus obtained was used as purified recombinant H. fusiformis cytochrome c 6. The degree of purity was confirmed by tricine SDS–PAGE (Schägger & von Jagow, 1987 ) and UV–visible spectroscopy. UV–visible spectra of H. fusiformis cytochrome c 6 were measured with a Hitachi U3310 spectrophotometer using quartz cuvettes of 1.0 cm path length. The concentration of the cytochrome c 6 was determined spectrophotometrically from the pyridine ferrohaemochrome spectrum (550 nm, 29.1 mM −1 cm−1). Potassium ferricyanide and sodium dithionite were used as the oxidant and the reductant, respectively.
The purified protein was dissolved in 10 mM sodium phosphate buffer pH 7.0 to prepare a concentrated protein solution of 20 mg ml−1. Initial crystals were obtained using the Wizard I random sparse-matrix crystallization screen (Emerald BioSystem). H. fusiformis cytochrome c 6 was crystallized by vapour diffusion using the hanging-drop method at 293 K. Each drop consisted of 2 µl protein solution and 2 µl reservoir solution. An initial crystal of H. fusiformis cytochrome c 6 grew within a week using condition No. 33 [2.0 M (NH4)2SO4, 0.1 M CAPS pH 10.5 and 0.2 M Li2SO4]. To improve the quality of the crystal, further screening for crystallization was performed and crystals were obtained reproducibly using 0.1 M CAPS pH 10.5, 0.2 M Li2SO4, 2.2 M (NH4)2SO4 and 3% glycerol. X-ray diffraction data were collected on BL-5A, Photon Factory, Tsukuba, Japan. The data set was processed with HKL-2000 and scaled with SCALEPACK (Otwinowski & Minor, 1997 ). The structure of H. fusiformis cytochrome c 6 was determined by molecular replacement using the program MOLREP (Collaborative Computational Project, Number 4, 1994 ). The search model used was Porphyra yezoensis cytochrome c 6 (Yamada et al., 2000 ). The structure of H. fusiformis cytochrome c 6 was refined with REFMAC from the CCP4 program suite. Water molecules were added using a water-pick script in CNS and refinement was continued using REFMAC5 (Collaborative Computational Project, Number 4, 1994 ). The final model obtained had an R factor of 18.4% and a free R factor of 20.9%. Manual model building was carried out using Coot (Emsley & Cowtan, 2004 ). Solvent molecules were placed at positions where spherical electron-density peaks were found above 1.5σ in the |2F o − F c| map and above 3.0σ in the |F o − F c| map and where stereochemically reasonable hydrogen bonds were allowed. A summary of the data-collection and refinement statistics is given in Table 1 .
To elucidate the genome code of a cytochrome c 6 gene from a brown alga, we determined the protein cDNA from the brown alga H. fusiformis as shown in Fig. 1 (Genbank accession No. AB105058). H. fusiformis cytochrome c 6 genes were amplified using cDNA, which was performed by the reverse transcription of poly(A)− mRNA. The polyadenylation signal sequences (AAUAAA) necessary for the addition of polyadenylic acid were not included in the 3′-region of the cytochrome c 6 gene from H. fusiformis, but the 3′-regions of the cDNA of the cytochrome c 6 that contained the sequence that can form a stem-loop structure that stabilizes mRNA were transcribed from the chloroplast genome (Drager et al., 1996 ; Yang & David, 1997 ). The gene that was transcribed from the chloroplast genome does not add polyadenylic acids (Sagher et al., 1976 ). Generally, the addition of polyadenylic acids that participate in mRNA stability occurs after transcription inside the nucleus (Darnell et al., 1971 ; O’Hara et al., 1995 ). The Shine–Dalgano (SD) sequence, a 16S-ribosomal RNA-binding site that is rich in purine 3–9 bases upstream of the initiation codon of prokaryotic cell mRNA (Bonham-Smith & Bourque, 1989 ), was present ten bases upstream of the initiation codon in the H. fusiformis cytochrome c 6 gene (Fig. 1 ). There were also no SD sequences in the cytochrome c 6 genes of the green alga C. reinhardtii (Merchant & Bogorad, 1987 ), the euglena Euglena gracilis (Vacula et al., 1999 ) and the cyanelle Cyanophora paradoxa (Steiner et al., 2000 ) that are encoded in the nuclear genome. We obtained a genomic DNA clone of approximately 730 bp that was amplified using primers constructed based on cDNA sequences (Fig. 1 ). The gene that encodes cytochrome c 6 was not inserted with an intron. The green alga C. reinhardtii gene encoding cytochrome c 6 has been reported to have its coding region interrupted by two introns (Hill et al., 1991 ). Genes encoded in nuclear genomes are usually inserted with introns (The Arabidopsis Initiative, 2000 ), but genes encoded in chloroplast genomes do not have these insertions (Shinozaki et al., 1986 ). These results showed that the cytochrome c 6 gene from the brown alga H. fusiformis was encoded in the chloroplast genome. At present, only red and brown algae have been reported to have a cytochrome c 6 gene encoded in the chloroplast genome.
E. coli BL21 (DE3) harbouring both pET22bHfc6 and pSTV28ccmA–H was used as a source of recombinant H. fusiformis cytochrome c 6. Recombinant H. fusiformis cytochrome c 6 was purified by ammonium sulfate pecipitation and two-step anion-exchange chromatography. The degree of homogeneity was confirmed by tricine SDS–PAGE and UV–visible spectroscopy. After initial purification by anion-exchange chromatography, tricine SDS–PAGE analysis displayed a predominant cytochrome c 6 band and a minor band and the fractions with an A 275/A 552.5 ratio lower than 2.0 were pooled and concentrated. After a second chromatography purification step, tricine SDS–PAGE analysis showed only the cytochrome c 6 band and the purification ratio (A 275/A 552.5) of H. fusiformis cytochrome c 6 was 0.90, which was similar to that of other cytochromes c 6.
The protein consists of 86 amino acids and one c-type haem and its molecular weight was calculated to be 9762.4 Da. From SDS–PAGE analysis a value of 8.0 kDa was obtained, which is somewhat lower than that deduced from the sequence (Fig. 2 a). Similar discrepancies have been observed in other small negatively charged proteins, such as the green alga Monoraphidium braunii cytochrome c 6 and plastocyanin (Campos et al., 1993 ).
The UV–visible spectra of reduced and oxidized recombinant H. fusiformis cytochrome c 6 are shown in Fig. 2 (b). In the reduced form, the α, β, γ (Soret) and δ absorption maxima peaks appear at 552.5, 522.0, 416.0 and 318.0 nm, respectively. For the oxidized form of the cytochrome c 6, the α + β and γ (Soret) absorption maxima peaks were 528.0 and 408.5 nm, respectively; a shoulder peak at 695.0 nm, indicating His–Fe–Met coordination, was observed (Fig. 2 b, inset).
A crystallization droplet was prepared by mixing 2 µl protein solution (20 mg ml−1 protein) in 10 mM sodium phosphate buffer pH 7.0 and 2 µl reservoir solution consisting of 0.1 M CAPS pH 10.5, 0.2 M Li2SO4, 2.2 M (NH4)2SO4 and 3% glycerol and was equilibrated against 500 µl of the same reservoir solution at 293 K. Diffraction-quality crystals appeared within a week (Fig. 3 ). This reservoir solution used for H. fusiformis cytochrome c 6 is somewhat similar to that used for cytochrome c 6 from the cyanobacterium Arthrospira maxima [reservoir solution containing 0.1 M Tris pH 7.8, 0.2 M Li2SO4, 2.2 M (NH4)2SO4 and 1% glycerol (Sawaya et al., 2001 )], but few similarities were found between the reservoir solutions used for H. fusiformis cytochrome c 6 and those used for other algal and cyanobacterial cytochromes c 6 (Kerfeld et al., 1995 ; Frazão et al., 1995 ; Schnackenberg et al., 1999 ; Yamada et al., 2000 ; Dikiy et al., 2002 ; Worrall et al., 2007 ).
The crystal structure of H. fusiformis cytochrome c 6 has been determined at 1.6 Å resolution. This is the first cytochrome c 6 crystal structure for a brown secondary symbiotic alga. The crystal belonged to space group P41212, with unit-cell parameters a = b = 84.58, c = 232.9 Å and six molecules (A, B, C, D, E and F) per asymmetric unit (Fig. 4 a). These six molecules could be superimposed with main-chain root-mean-square deviation (r.m.s.d.) values of 0.1–0.4 Å, as determined using the DALI program (Holm & Park, 2000 ). The hexamer contains four sulfate ions which may be derived from the ammonium sulfate and lithium sulfate included in the crystallization solution. The cytochrome c 6 hexamer was formed of a dimer of trimers (ABC and DEF trimers; Fig. 4 b). An intermolecular hydrogen bond was formed in the ABC trimer between each pair of molecules in the trimer through Ala60 N and Arg64 O and one sulfate ion was centred between the Arg64 side chains of the three molecules (Fig. 4 c). This arrangement of a sulfate ion enclosed by a basic amino-acid residue has been also found in the crystal structure of Hydrogenobacter thermophilus cytochrome c 552 (Travaglini-Allocatelli et al., 2005 ). Considering that the crystals of H. fusiformis cytochrome c 6 were obtained in the presence of sulfate ions, the sulfate ions were convenient for crystallization and might contribute to crystal-packing stabilization by neutralization of charge repulsion in this region. Therefore, we deduce that the crystallographic hexamer is a nonphysiological crystal-packing artifact. An intermolecular hydrogen bond was formed between each pair of molecules of the second trimer through the C-terminal Asn86 Nδ2 and Asn86 OX (Fig. 4 c). Hydrogen bonds between the two trimers were formed between Asn22 Oδ1 of the ABC trimer and Arg64 N2 οf the DEF trimer.
An oligomeric arrangement of molecules has been found in the crystal structures of other cytochromes c 6. A trimeric arrangement of molecules has been found in the structures of cytochrome c 6 from C. reinharditii form I (Kerfeld et al., 1995 ) and M. braunii (Frazão et al., 1995 ). The proteins from Scenedesmus obliquus (Schnackenberg et al., 1999 ), A. maxima (Sawaya et al., 2001 ) and Phormidium laminosum (Worrall et al., 2007 ) have been crystallized as dimers. These oligomers of cytochromes c 6 were formed by the packing of different molecules and were not superimposed. It has been reported that the observed differences in oligomerization between various cytochromes c 6 may be determined by subtle differences in their surface electrostatic potential properties (Dikiy et al., 2002 ). In contrast, the cytochromes c 6 from P. yezoensis (Yamada et al., 2000 ) and Cladophora glomerate (Dikiy et al., 2002 ) are monomeric in the crystal.
The structure of H. fusiformis cytochrome c 6 belongs to the class I c-type cytochromes, which are composed of four α-helices and tight turns (Fig. 5 ). The protein consists of a single polypeptide chain folded around the haem prosthetic group. The secondary structures have been classified according to the criteria of Kabsch & Sander (1983 ). Four α-helices, Asp2–Asn13 (I), Ser15–His18 (I′), Lys33–Ala38 (II), Ile44–Asn53 (III) and Asp67–Lys83 (IV), are found as elements of a regular secondary structure, with helices I and IV overlapping at about 90° (Fig. 3 ). A two-stranded antiparallel β-sheet was formed with two interchain hydrogen bonds between Lys55 and Met58, which form a type II′ β-turn with Asn56 and Ala57. A short β-sheet has commonly been observed in the structures of cyanobacterial, green and red algal cytochromes c 6.
The crystal structures of four cytochromes c 6 from the eukaryotic green algae C. reinharditii (Kerfeld et al., 1995 ), M. braunii (Frazão et al., 1995 ), S. obliquus (Schnackenberg et al., 1999 ) and C. glomerata (Dikiy et al., 2002 ), of one from the eukaryotic red alga P. yezoensis (Yamada et al., 2000 ) and of two from the prokaryotic cyanobacteria A. maxima (Sawaya et al., 2001 ) and P. laminosum (Worrall et al., 2007 ) have been determined. They are composed of 85–90 amino acids and their main secondary-structural elements are α-helices wrapping around the haem prosthetic group. An amino-acid sequence comparison of H. fusiformis cytochrome c 6 with those from C. reinharditii, M. braunii, C. glomerata, S. obliquus, P. yezoensis, A. maxima and P. laminosum revealed similarities of 46.67, 47.78, 45.05, 47.19, 72.09, 53.33 and 59.77%, respectively (Fig. 6 ) and the amino-acid sequence of H. fusiformis cytochrome c 6 is most similar to that of P. yezoensis cytochrome c 6. The main-chain r.m.s.d.s between H. fusiformis, C. reinharditii, P. yezoensis and A. maxima cytochromes c 6 are 0.5–1.1 Å, as determined using the DALI program (Holm & Park, 2000 ). A Cα trace of H. fusiformis cytochrome c 6 shows a high overall similarity between the green algal and cyanobacterial cytochromes c 6, as well as subtle differences (Fig. 7 ). The largest deviation in the Cα trace between the brown alga H. fusiformis cytochrome c 6 and green algal and cyanobacterial cytochromes c 6 was found in the second interconnecting loop (Gln40–Ser43; Fig. 7 ). The green algal and cyanobacterial cytochromes c 6 have a small insertion of 2–4 amino acids in this region compared with H. fusiformis cytochrome c 6. The loop region in H. fusiformis cytochrome c 6 resembles that in cytochrome c 6 from the red alga P. yezoensis, which also lacks two amino acids in this region compared with green algal and cyanobacterial cytochromes c 6. Considering that the loop region of cytochromes c 6 has a poorly conserved amino-acid sequence compared with other regions, this region may have no common biological functional role. In the structure of other cytochromes, functional roles have not been reported for this region.
In this study, we showed that the cytochrome c 6 gene from the brown alga H. fusiformis was encoded in the chloroplast genome. To date, the cytochrome c 6 gene has only been found to be encoded in the chloroplast genome in red and brown algae. The amino-acid sequence and tertiary structure of H. fusiformis cytochrome c 6 were very similar to those of a red algal cytochrome c 6 rather than those of green algal cytochromes c 6. The present results support the hypothesis that brown algae gained their chloroplasts via secondary endosymbiosis involving a primitive red algal endosymbiont and a nonphotosynthetic eukaryote host.
We thank Messrs Daisuke Tamura, Naoya Terunuma and Masaki Hosokawa and Mses Ayako Ohsuzu, Ayumi Hisamitsu and Kasumi Suzuki of Nihon University for the expression and purification of H. fusiformis cytochrome c 6. This work was supported in part by a Nihon University Multidisciplinary Research Grant for 2008.