Purification and Spectroscopic Characterization of a Recombinant ...

Eur. J. Biochem. 242, 779-787 (1996) 0 FEBS 1996

Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos Manon COUTURE and Michel GUERTIN Department of Biochemistry, Laval University, Quebec, Canada (Received 31 July 1996) - EJB 96 1138/3

Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast. In order to understand the role played by this new Hb, we have undertaken its biochemical characterization. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01 % of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant protein possesses a non-covalently bound iron-protoporphyrin IX heme. The oxy form of the recombinant Hb, purified directly from bacterial cells, is very stable, with a measured half-life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the LI637 Hb can be reduced by the electron-transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules. With regard to this, we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form. Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP reductase are used to enzymically reduce the Hb. Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low-spin at physiological pH. These results confirm the existence of a reversible oxygen-binding protein in the chloroplast of C. eugametos. The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb. +

Keywords: hemoglobin ; Chlamydomonas ; green alga; oxygen-binding protein.

Two groups of hemoglobins (Hbs) have been identified so far i n unicellular organisms. The first group includes the dimeric Hb of the filamentous purple bacterium Vitreoscilla (VtHb; Wakabayashi et al., 1986) as well as the flavohemoglobins (FHbs) found in a variety of purple bacteria (Cramm et al., 1994; Favey et al., 1995; Vasudevan et al., 1991) and fungi (Iwaasa et al., 1992; Zhu and Riggs, 1992). In FHbs, the heme-binding domain is found in the amino-terminal portion of the protein and shares significant sequence similarity to almost all VtHb (39-46% amino acid identity) ; the flavin reductase activity is associated with the carboxyl-terminal domain. Phylogenetic analyses indicate that the distribution of these Hbs is best explained by a horizontal gene transfer event in which a common ancestor of Vitreoscilla, Escherichia coli and Alcaligenes eutrophus acquired a copy of the FHb gene from an ancestor of SacchuroCorrespondence to M. Guertin, Department of Biochemistry, Marchand Building, room 3145, Laval University, Quebec, Canada, G1K 7P4 Fax: + I 418 656 7176. Ahhreviations. Hb, hemoglobin; Mb, myoglobin; FHb, flavohemoglobin ; IPTG, isopropylthio-/I-galactoside;P,,,,half-saturation oxygen tension.

myces cerevisiae soon after this latter diverged from an ancestral Candida nowegensis (Moens et al., 1996). According to this hypothesis, the flavin reductase domain was lost in the Vitreoscilla lineage. The recently solved crystal structure of A. eutrophus FHb (Ermler et al., 1995) shows that the heme-binding domain has retained the classical globin fold, although the D helix is absent. However, marked differences were observed in the conformation and environment of the proximal histidine residue, which is hydrogen bonded to a glutamine residue and a tyrosine residue. The latter glutamine residue is also hydrogen bonded to the tyrosine residue and to a valine residue via a water molecule. Such an arrangement may confer interesting electrontransfer properties to the protein. The second group of Hbs comprises those identified in the ciliated protozoa Tetrahymena and Paramecium (Iwaasa et al., 1989; Takagi et al., 1993; Yamauchi et al., 1995), the cyanobacterium Nostoc commune (cyanoglobin) (Potts et al., 1992) and the green unicellular alga Chlamydomonas eugametos (Couture et al.., 1994). Comparative protein sequence analysis revealed that these Hbs are highly related to each other but do not show significant similarity to other globins, except for the invariant residues associated with the oxygen-binding properties (Couture

7 80

Couture and Guertin ( h i : J. Biochern. 242)

et al., 1994). However, their respective sequences match well to the Bashford template, based on the alignment of vertebrate and non-vertebrate Hb sequences (Bashford et al., 1987) as well as to a new template based solely on non-vertebrate Hb sequences (Moens et al., 1996). This indicates that the members of this second group of Hbs are true globins and should possess the globin fold. Phylogenetic analyses have confirmed their close relationship and have suggested a possible evolutionary scheme in which cyanobacteria acquired the Hb gene by horizontal gene transfer from a common ancestor of Parumecium, Tetrahymena and Chlamydomonus (Moens et al., 1996). Little is known about the biochemical properties of the Hbs of the latter group and their function remains to be elucidated. Paramecium caudatum Hbs are monomers which bind oxygen with high affinity (half-saturation oxygen tension of 0.6 mm Hg) (Smith et al., 1962) and autoxidize relatively rapidly (Tsubamoto et al., 1990). I n vivo, they are detected in cultures in the logarithmic and stationary growth phases (Usuki and Hino, 1991). The cyanoglobin also binds oxygen reversibly with half-saturation oxygen tension (Ps,,) values of 0.32-0.99 mm Hg (Thorsteinsson et al., 1996). It is detected only under microaerobic conditions when the cells are starved of fixed nitrogen, suggesting that it could be involved in some aspect of nitrogen fixation (Potts et al., 1992). Interestingly, the gene encoding cyanoglobin (GIDN) is located between two genes that are essential for nitrogen fixation. In C. eugametos, the putative Hbs are encoded by a small gene family, synthesized on cytoplasmic ribosomes and exported to the chloroplast where they are found distributed between the pyrenoid and the thylakoid regions (Couture et al., 1994). Two members of this gene family, LZ637 and LZ410, are periodically expressed in response to illumination and the activation of photosynthesis (L1637) in cells grown under light/dark regimes (Couture et al., 1994; Gagne and Guertin, 1992). To facilitate the biochemical characterization of Chlamydomonas Hbs, the L1637 protein was expressed in E. c d i . The recombinant protein was purified to apparent homogeneity and its different oxidation states were investigated. We show here that the recombinant protein can reversibly form a very stable complex with oxygen, supporting its designation as a hemoglobin.

MATERIALS AND METHODS Materials. Mes, Mops, Taps, Ches, Caps and Tris used for buffer systems were obtained from ICN, ammonium sulfate was from Gibco BRL, while the chemicals and biochemicals used for enzymic and non-enzymic reduction of Hbs were from Sigma. Molecular biology products were from varied sources. The Pfu DNA polymerase was from Stratagene, restriction enzymes were from Pharmacia Biotech, DNaseI was from Gibco BRL, the Magic PCR Preps DNA was from Promega Corporation, the Transformer Site-Directed Mutagenesis kit was from Clontech and the dideoxy sequencing kit (Sequenase version 2.0 T7 DNA polymerase) was from USB. The Western-blot chemiluminescent system was from Dupont NEN. Carbon monoxyde gas (UHP grade) was purchased from Air products. Plasmids construction. Specific segments of the LI637 cDNA were cloned i n the prokaryotic expression vector pET3A (Novagen) to produce recombinant LI637 proteins. The desired sequences were amplified by PCR with primers derived from the LI637 cDNA sequence. A 13-nucleotide sequence containing a NdeI restriction site overlapping an appropriately positioned initiation codon was added to the upper (5’) primers. The upper primers used were (5’43 CTG TTC CAT ATG TCG CTC TTC GCC AAG CTG-3’) for the H19 clone (positions 212-229 in

the LI637 cDNA; Fig. 2B in the reference (Couture et al., 1994)) and (S’-G CTG TTC CAT ATG ACG TCC GCC ACT CCC GCC-3’) for the H20 clone (positions 155-172). The lower primer (5’-ATC CGG CTG CTG CTC TCC ACA CC-3’), localized at positions 706-723 bp, was used in combination with the H19 and H20 PCR primers. The DNA amplifications were performed in a final volume of 50 pl containing 20 mM Tris/CI, pH 8.75, 10 mM KCI, 10 mM (NH,&S04, 2 mM MgSO,, 0.1% Triton XI00 and 0.1 nig/nd BSA with the use of 1.25 U Pfu DNA polymerase and 0.1 ng LI637 cDNA for 30 cycles, including a l-min denaturation step at 94”C, a 25-s annealing step at 55°C and a 3-min elongation step at 72°C. The amplified fragments were purified using the Magic PCR Preps DNA purification system (Promega), digested at the NdeI and BclI unique restriction sites and cloned at the NdeI and BamHI sites of the vector pET3A. The BclI site is not provided by the lower PCR primer; this restriction site is found at the position 672 in the L1637 cDNA. The sequence of the cloned fragments was verified by dideoxy sequencing. Site-directed mutagenesis. The cysteine residue found at position 41 in the H20 protein (Fig. 1 a) was changed to a lysine residue by the site-directed mutagenesis procedure of Deng and Nickoloff (Deng and Nickoloff, 1992) with the use of the Transformer Site-Directed Mutagenesis kit from Clontech. The mutagenic primer used (S’-CGA CGA CGG CTT CTT CCT AGC C3’) is located at nucleotides 193-214 on the non-coding strand of LJ637 cDNA, while the selection primer (S’-GTG ACT GGT GAG GCC TCA ACC AAG TC-3’) is located at nucleotides 4113-4138 in the pET3A vector. The latter primer changed a unique ScaI restriction site to a unique Stul restriction site in the ampicillin-resistance gene. This new clone, termed H21, was resequenced by the dideoxy method. Expression and purification of recombinant protein. E. coli cells [strain BL21 (DE3)], containing the various plasmids, were grown overnight at 37°C in a New Brunswick incubator set at 200 rpm in 250-mI flasks containing 125 ml Luria-Bertani medium supplemented with 50 pg/ml ampicillin. These cultures were used to seed 2-L flasks containing 500 ml fresh Luria-Bertani media and 50 pg/ml ampicillin (1 :100 dilution factor). Cells were grown as before to an A,,,,,,,,,, of 0.6-0.8, at which point they were incubated for an additional 2 h either in the presence or absence of 0.4 mM isopropylthio-/j-galactoside (IPTG). The cells were harvested by centrifugation at 5000 g for 10 min at 4”C, resuspended in 0.02 vol. buffer containing SO mM Tris/CI, pH 7.5. SO pM EDTA and 1 mM phenylmethylsulfonyl tluoride and disrupted by two passages in a French pressure cell operated at 137 MPa (cooled at 4°C). DNasel was added to a final concentration of 1 pg/ml and cell debris were removed by centrifugation at 12000 g for 30 min at 4°C. The extract was then fractionated with ammonium sulfate at 40-80% saturation. The precipitate was collected by centrifugation at 10000 g for 20 min, resuspended in 10 mM Tris/CI, pH 8, SO pM EDTA and dialysed against the same buffer overnight at 4°C in 6-8-kDa molecularmass cutoff membranes from Spectrapor. The dialysate was then loaded onto a 2.5 cmX30 cm column of DEAE-Sephacel (Pharmacia), equilibrated with 10 mM Tris/ CI, pH 8, 50 pM EDTA at 4°C (14-17 mg protein/ml resin). The column was washed with the same buffer until the A,,,,,,,,, was less than 0.05. The recombinant Hb was then eluted with 10 mM Tris/Cl, pH 7.5, 50 pM EDTA buffer containing either 25 mM NaCl (H20 and H21 proteins) or S O mM NaCl (HI9 protein). The fractions containing the Hb, identified by measuring the absorbance at 410 nm and 280 n m and by Western-blotting analysis, were concentrated by ammonium sulfate precipitation (90% saturation). The proteins were then resuspended in 1-2 ml buffer containing 50 mM Tris/CI, pH 7.5, SO pM EDTA

Couture and Guertin (Eur: J . Biochem. 242)

and 100 mM NaCl, dialysed against the same buffer overnight at 4"C, and passed through a HiLoad 16/60 Superdex 75 gelfiltration column (prep grade, Pharmacia) equilibrated with the same buffer at room temperature. Colored fractions containing recombinant Hb were concentrated by ammonium sulfate precipitation, as described previously, and were dialysed against 10 mM Tris/Cl, pH 7.5, SO pM EDTA overnight at 4°C. The Hb (1-2 mg) was then loaded onto a Resource Q 1 ml column (Pharmacia) at a flow rate of 2 ml/min and eluted with a linear gradient (40 ml) of 0-100 mM NaCl in 10 mM Tris/Cl, pH 7.5, SO pM EDTA at room temperature. This last chromatography step eliminates remaining contaminants and allows the separation of the met and oxy forms. Purified recombinant Hbs were concentrated to 0.5 - 1 mg/ml by ultrafiltration (Spin-X Uf, 10kDa molecular-mass cutoff; Costar) and kept at 4°C until use. The purified oxy form was used immediately while the met form was used within 2 days following purification. The purity of each fraction was assessed by SDS/PAGE (5 % stacking/lS % resolving gel) and Coomassie brilliant blue R-250 or Silver nitrate staining. Protein concentrations were determined by the Bradford method (Bio-Rad) using BSA as a standard. The heme was identified and quantified by the alkaline pyridine method as described by Poole et al. (1986) and Appleby ( 1978). Protein analysis. The molecular masses of the recombinant Hbs were also determined by gel-filtration chromatography following calibration of the Superdex 75 gel-filtration column (Pharmacia) with a mixture of molecular-mass standards (gelfiltration standards, Bio-Rad). Western-blot analyses were performed as previously described (Couture et al., 1994) except that a chemiluminescent detection system (Renaissance, Dupont NEN) was used in conjunction with anti-rabbit-IgGs-coupled horse radish peroxidase (Amersham Life Science). Heme staining of SDS/PAGE (Francis and Becker, 1984) was performed using 3,3'-diaminobenzidine (Sigma). Spectrophotometric studies. Absorption spectra were recorded with a Cary 3E U/V visible spectrophotometer (Varian) equipped with a thermostatically controlled multicell holder set at 23°C. The heme content quantified by the pyridine hemochrome method (Appleby, 1978) was used to calculate millimolar absorption coefficients for each derivative. Spectra of the met form at various pH values were collected following mixing of the met protein, in 3 mM Tris/CI, pH 7.5, with 1 vol. of the following buffers (50 mM) : sodium acetate, pH 5 ; Mes/NaOH, pH 6 ; Mops/NaOH, pH 7 ; TapdNaOH, pH 8 ; Ches/NaOH, pH 9 ; Caps/NaOH, pH 10. The cyanide and azide derivatives were obtained following the addition of either 2 p1 0.5 M potassium cyanide or 5 p1 1 M sodium azide to 1 ml hemoprotein. Hb was reduced non-enzymically by the following manipulations in an anaerobic chamber (Labmasterloo, MBraun) using deoxygenated buffers. The recombinant protein was reduced with a few grains of solid sodium dithionite. The solution was then passed through a 3-ml Bio-Gel P6DG column (Bio-Rad) equilibrated with 10 mM Tris/Cl, pH 7.5, containing 50 pM EDTA. The protein was diluted to a final volume of 3 ml in a sealed cuvette (Hellma) and its spectrum was immediately recorded. The spectrum of the oxy form was recorded after exposing the content of the cuvette to air and mixing by inversion. Hb was reduced enzymically using ferredoxin and ferredoxin NADP' reductase as described by Hayashi et al. (1973). Recombinant Chlamydomonas Hbs (5-6 pM in heme) and horse heart Mb (4 pM) were incubated in a buffer containing 45 pM NADP, 670 pM glucose-6-phosphate, 0.13 U/ml glucose-6-phosphate dehydrogenase (type XII from Torula yeast), 0.17 pM spinach ferredoxin, 3.1 pg/ml spinach ferredoxin NADP' reductase,

781

1.7 pg/ml bovine liver catalase, 10 mM Tris/Cl, pH 7.5, and 50 pM EDTA. The reactions were performed at 23°C in the cuvettes and were initiated by the addition of glucose-6-phosphate dehydrogenase. Spectra were recorded at various time intervals after the initiation of the reaction. Hb was also reduced, using phenazine methosulfate as the electron carrier, as described by Kajita et al. (1970) except that NADPH was used in place of NADH. Recombinant Chlamydomonas Hbs (5-6pM in heme) and horse heart Mb (4pM in heme) were incubated in 10 mM Tris/Cl, pH 7.5, containing SO pM EDTA, 1 mM NADPH, 0.2.5 pM phenazine methosulfate and 1.7 pg/ml bovine liver catalase at 23 "C. The reactions were initiated by the addition of NADPH and the spectra were recorded at various time intervals after the initiation of the reaction. The carbonmonoxy complex was obtained following equilibration with CO. Autoxidation. The autoxidation rate of recombinant Hb was measured in 2.5 mM buffers at pH 5-10 in quartz cuvettes sealed with teflon stoppers at 23°C. 1 vol. 50 mM buffer was mixed with 1 vol. oxy Hb (7 pM) and the spectra (250-700 nm) were recorded at 10-min intervals (pH 5 ) and 30-min intervals (pH 6-10). A few grains of solid potassium ferricyanide were added to record the final met spectra. The buffers used were sodium acetate, pH 5 , Mes/NaOH, pH 6, Mops/NaOH, pH 7, TapsNaOH, pH 8, Ches/NaOH, pH 9 and Caps/NaOH, pH 10. The autoxidation rate (Tsubamoto et al., 1990) was calculated from the absorbance change at 581 nm (cx-peak).

RESULTS Expression and purification of recombinant Chlamydomonas Hb. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01% of the soluble protein in the green alga, we chose to purify a heterologously expressed protein. To enable its expression in E. coli, the LI637 cDNA, encoding one of the putative Chlamydomonas Hbs, was subcloned into the pET3A expression vector. As observed for other nuclear-encoded polypeptides, the LI637 protein possesses an amino-terminal transit peptide which serves to target the apoprotein to the chloroplast. Once inside the chloroplast, the transit peptides are usually cleaved and eliminated. Although all transit peptides share common features, it is still impossible to accurately predict the cleavage site by inspection of the amino acid sequence (Keegstra, 1989). A recombinant protein whose size reflects that of the mature Chlamydomonus Hb (16 kDa) was created by removing the first 24 amino acids of the LI637 protein. The first residue of this recombinant protein, termed H20, was thus Thr25 of the wildtype Hb (Fig. la). H20 migrated with an apparent molecular mass close to that of native Chlumydonzonas Hb, as determined by SDSPAGE and Westem-blot analysis (Fig. 1b). During its purification, the H20 polypeptide showed a strong tendency to form dimers. While the addition of dithiothreitol or 2-mercaptoethanol to the purification buffers prevented this dimerization, the use of these compounds proved undesirable as each formed complexes with the met form of H20 (Table 1). H21, a variant of H20, was created by substituing the unique cysteine residue found at position 41 with a lysine residue. This residue is found in the corresponding position in Tetruhymena pyriformis Hb. The recombinant H20 and H21 Hbs were produced when the cloned genes were placed under the control of the T7 RNA polymerase promoter in the pET3A expression vector. The addition of isopropylthio-0-gaiactoside had no effect on the level of recombinant protein expression. Attempts to improve the yield

Couture and Guertin (Eur: J. Biochem. 242)

782

A H19

H20, H21

4

C.eug. LI637 MMRTVQLRTL RPCIRAQQQP VRPSTSATAA AATAPAPARK C.e u g . Li410 MMRTVQLRTL RPCIRAQQQP VRAPTSVAAA TATTPAPTKK __________ __________ __________ P.cau. B T.pyr. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ MN __________ __________ __________ __________ N.corn.

__________

4

CPSSLFAKLG GREAVEAAVD CPFSLFAKLG - - - SLFEQLG KPQTIYEKLG -MSTLYDNIG

GREAVEAAVD GQAAVQAVTA GENAMKAAVP GQPAIEQVVD

66245'0-

3L'021.5. L4,4-

C

45.0.

45.0.

31.0.

31.0-

21.5.

21.5.

14.4-

14.4-

6.5.

6.5-

Trmc (mimics)

Fig. 1. Expression and purification of recombinant Chlumydomonus Hbs. (a) Alignment of the amino-terminal portions of the Hbs of C. eugumetos ILI637, GenBank accession number X72916 and LI410 (X72Y15)], P caudutum (D12916), 7: pyrijbrmis (D13920) and N . commune (MY2437).

The arrows denote the first amino acid following the initiating methionine; Thr25 for the H20 protein, Ser44 for the H1Y protein. The H21 protein is the [Cys41Lys] variant of H20 (see text for details). (b) Western-blot analysis of the purified H20 protein and of C. eugarnetos soluble protein extract (10 pg). (c) Elution profile of the H21 protein on a Resource Q column with a linear NaCl gradient. (d) Analysis of the purified Hbs by SDSlPAGE and Coomassie blue staining (1-2 pg purified proteins/lane). (e) Heme staining of the H20 protein, horse heart Mb and cytochrome L' following SDSlPAGE (100 pmol hemellane).

(1-2 mg/l) by varying the temperature of growth and supplementing the media with 8-amino levulinic acid and trace elements were unsuccessful. The recombinant proteins were purified to near homogeneity (Fig. I d ) by a four-steps procedure which includes ammonium sulfate fractionation, anion-exchange chromatography (DEAESephacel), gel-filtration chromatography (Superdex 75) and a final anion-exchange chromatography (Resource Q) which resolves the met and oxy forms (Figs Ic, 2a). The recombinant H21 protein was a monomer by gel-filtration chromatography (result not shown). Heme staining following SDSPAGE indicates that the heme is non-covalently bound to the protein (Fig. 1e) while the dithionite-reduced minus oxidized spectrum of the pyridine hemochrome complex indicates that the E. coli-derived heme is protoporphyrin IX (Table 1 ; Appleby, 1978).

The H21 protein hinds oxygen and carbon monoxide. Two fractions containing the H21 Hb were eluted from the final QAE chromatography column (Fig. 1 c). The first fraction, eluted at a salt concentration of 14 mM, has an ultraviolet/visible spectrum closely resembling those of oxyMbs and oxyHbs, with a Soret band positioned at 412 nm, an a peak at 581 nm and a p peak at 545 nm (Fig. 2a). The Soret band is blue-shifted as compared to most Hbs, as has been observed for some Hbs such as the leghemoglobin of Myricu gale which has a Soret peak at 41 1 nm (Pathirand and Tjepkema, 1995) and the Hb of Ascuris (Soret band at 4 1 2 n m ; Darawshe et al., 1987). The spectrum of the oxy H21 protein is also very similar to those of I? caudutum, 7: pyrijorrnis and N . commune Hbs in that the a peak is less intense than the , ! Ipeak (a/pn,ux peak ratio of 0.75). In contrast, the alp,,,,, peak ratio of vertebrate oxyMbs and oxyHbs is approximately 1.05. Equilibration of this first fraction with carbon monoxide gas yields a form whose spectrum is characteristic of a carboninonoxy complex, indicating that it indeed contains a heme pro-

tein in the reduced state (Fig. 2b). Finally, the addition of sodium dithionite to the oxy form converts it to the deoxy form (Fig. 2 b), while the addition of potassium ferricyanide converts it to the met form (Fig. 2a). The dithionite-reduced Hh has an unusual spectrum in the visible region, having a well resolved peak at 557 nm and a shoulder at 529 nm (Fig. 2b). This spectrum resembles that of reduced cyanoglobin (Potts et al., 1992). The peak at 557 nm is reflected in the CO-difference spectrum as a trough at 557 nm (Fig. 2 b , inset). However, the intensity of the trough is not as pronounced as those observed for proteins having a low-spin heme, suggesting that the heme of reduced Chlurnydomorzas Hb is present as a mixture of low-spin and high-spin states according to the criteria of Wood (Wood, 1984). A true low-spin form in the reduced state can be obtained midazole is added to thc met form prior to the reduction with dithionite. The spectrum thus obtained, with peaks at 427, 529 and 557 nm, is very similar to those of b-type cytochromes such as the cytochrome b, (result not shown; Beck von Bodman et al., 1986).

Spectra of the met H21 protein. The spectrum of the met form of the recombinant H21 protein at pH7.5 is characterized by two peaks at 410 nm and at 535 nm (Fig. 2a, Table 1). The spectrum differs from those of the high-spin aquo met form or low-spin alkaline met form of Mhs and Hbs hut closely resembles those of hemichromes (low-spin hexacoordinate heme) in which the sixth coordination of the iron atom is provided by an endogeneous amino acid. The spectrum of the met form of the H21 protein is very similar to the putative hemichrome of f? caudatum Hb, which is characterized by a peak at 538 nm and a shoulder at 560 nm (Tsubamoto et al., 1990). The formation of the latter hemichrome is observed after the autoxidation of the oxy form at alkaline pH or after long-term storage of the met form at neutral pH. The met H21 protein spectrum is also similar to the spectrum of the met cyanoglobin at neutral

Couture and Guertin ( E m J . Biochem. 242)

783

0.8

0.1

0.7

0.09 0 08

0.6

0.07

0.5

A

A

0.06

0.4

0.05

0.3

0.04 0.03

0.2

0.02 0.1

0.01

0

0

250 300 350 400 450 500 550 600 650 700

Wavelength (nm)

400

450

500 550 600 650 700

Wavelength (nm)

Fig. 2. The absorption spectra of the purified H21 protein. (a) Spectra of the oxy form (-) and of the met form (- - -). Protein concentration was 7.7 pM, based on the heme content. (b) Spectra of the dithionite reduced (---) and carbonmonoxy forms (-). The inset shows the reducedplus-CO minus-reduced difference spectrum. The Hb concentration was 4 pM. Spectra were recorded in 10 mM Tris/CI, pH 7.5, at 23°C.

0.6 I

0.5

A

0.4

04

0.04

03

0.03

0.2

0.02

0.1

0.01

0+ 250 300 350 400 450

4

0 550 600 650 700

Wavelength (nrn)

300 350 400 450 5M) 550 600 650 700

Wavelength (nm)

Fig.3. The pH dependence of the absorption spectrum of the met form of the H21 protein. Spectra at pH 5-8 were recorded in 25 mM buffers at 23°C. The buffers used were sodium acetate, pH 5, Mesl NaOH, pH 6, Mops/NaOH, pH 7, and Taps/NaOH, pH 8. The Hb concentration was 3 pM.

Fig.4. The metH2l Hb complexes with azide and cyanide. (-) The met form of the H21 protein in 10 mM Tris/CI, pH 7.5; (- . ) cyanide complex obtained after the addition of KCN to a final concentration of 1 mM; (. . . ' .) azide complex obtained after the addition of NaN, to a final concentration of 5 mM. The Hb concentration was 3.8 pM.

pHwhich has a peak at 540nm and a shoulder at 570nm (Thorsteinsson et al., 1996). No change occurs to the spectrum of met H21 Hb when the pH is changed from 8 to 10 (result not shown). However, as the pH was lowered, a peak appeared at 624 nm (Fig. 3). This peak is typical of high-spin hexacoordinated metHbs, thus indicating a change in the spin state. Therefore, the low-spin form present at neutral and alkaline pH values is converted to a high-spin hexacoordinate form at acidic pH following the protonation of the distal amino acid ligand or a conformational change of the protein.

Formation of the oxy complex in vitro. At the physiological pH of the chloroplast (pH 7-8), Chlamydomonas Hb should be found in the low-spin hexacoordinate form when the heme is oxidized. To demonstrate that this met form is still a functional protein able to bind oxygen, three approaches were used to convert it to the oxy form in vitro. As a first attempt, the purified met H21 protein was reduced with sodium dithionite and was purified from excess reagents by gel-filtration chromatography to allow oxygenation. This procedure failed to oxygenate the protein and resulted in the conversion of the deoxy form to the met form, although the horse heart met Mb used as a control was quickly converted to the oxy form under identical conditions (result not shown). In contrast, when the reduction and excess reagent removal were performed in an anaerobic atmosphere, the deoxy form was fully oxygenated by mixing with air (Fig. 5 ) , indicating that the reduction and excess salt removal must be performed in the absence of oxygen. The oxy form could, nevertheless, be obtained under an atmosphere of oxygen with the use of NADPH and electrontransfer mediators. In a reducing system containing a NADPHgenerating system, the electron-transfer mediators ferredoxin and ferredoxin NADP' reductase, as well as catalase to destroy hydrogen peroxide (Hayashi et al., 1973), the met H21 protein was rapidly converted to the oxy form (< 15 min) and was stable

Cyanide and azide binding. Although the heme of the met H21 protein is probably low-spin and six-coordinate at pH 7.5, it can bind anionic ligands such as cyanide and azide, forming complexes that have spectra characteristic of other Hbs. The spectrum of the cyanide complex at pH 7.5 has peaks at 415 nm and 540 nm (Fig. 4, Table 1) while that of the azide complex has peaks at 414 nm and 546 nm with a shoulder near 580 nm (Fig. 4). Identical spectra were obtained at pH 5 (result not shown). However, the met H21 Hb does not bind fluoride at neutral and acidic pHs even in the presence of a large excess of this weak field ligand (20000-fold molar excess over heme; data not shown).

Couture and Guertin (Eur: J. Biochern. 242)

7 84

00.1

0 03

0 02

0 01

0

250 300 350 400 450 500 550 600 650 700

the H21 protein was incubated in the presence of NADPH and phenazine methosulfate. The oxy form was converted to the met form within a few minutes after the addition of NADPH (Fig. 6 c). This NADPH-mediated conversion of the oxy form to the met form requires the presence of an electron-transfer mediator, as it does not take place in the absence of phenazine methosulfate. Moreover, the reaction is not the result of a direct oxidation by phenazine methosulfate itself as it does not occur i n the absence of NADPH (result not shown). The latter results suggest that the dioxygen molecule of the oxy H21 protein is reactive in the presence of an electron-donor compound and the small electron carrier phenazine methosulfate, but not in the presence of the ferredoxidferredoxin NADP reductase couple. The oxy H21 protein was also stable in the presence of other electrondonor compounds such as reduced glutathione, ascorbate, dithiothreitol and 2-mercaptoethanol. +

Wavelength (nm) Fig.5. In vitro oxygenation of the H21 protein following reduction and desalting under a nitrogen atmosphere. Absorption spectra of the dithionite-reduced H21 protein after excess salt removal under il nitrogen The Hb atmosphere (---) and after opening the cuvet to air (--). concentration was 2.5 pM.

for at least 1 h (Fig. 6a). As previously observed for metHb, ferredoxin was required for this conversion to take place. Not all electron carriers were found to be effective, however. For instance, when phenazine methosulfate (Kajita et al., 1970) was substituted for the ferredoxidferredoxin NADP' reductase couple, no apparent reduction of the met H21 protein was observed, even after prolonged incubation (result not shown). However, when the reaction was performed under an atmosphere of carbon monoxide, the carbonmonoxy complex was formed within 30-40 min, indicating that the heme was indeed reduced by the phenazine methosulfate/NADPH couple (Fig. 6 b). In contrast, under an oxygenic atmosphere, horse heart metMb was rapidly and stably converted to oxyMb in the phenazine methosulfate/NADPH reducing system (Fig. 6 d). Under these conditions, the oxyMb was formed within a few minutes and was stable for at least 1 h. These results suggest that, under an oxygenic atmosphere, the oxy form of the H21 protein was probably formed but was unstable. To demonstrate this, the oxy form of

Autoxidation. The Hbs of P: caudatum and N. commune autoxidize relatively rapidly i n comparison to various vertebrate Mbs. The apparent half-life of P. caudatum Hb at pH 9, the pH optimum of the stability of the oxy complex, is =63 h (k,,,,, = 0.011 h '; Tsubamoto et al., 1990) while that of the cyanoglobin is less than 12 h (Thorsteinsson et al., 1996). In contrast to these Hbs, the oxy form of the H21 protein was more stable, with a measured half-life of 7 days at pH 8 (kc,,, = 0.0041 h ' ; Fig. 7). The half-life of horse heart Mb measured under the same conditions was 4.8 days (k,,,, = 0.006 h-'; results not shown). As expected, the autoxidation rate of the H21 protein was found to be pH dependent. The longest half-life was measured at pH 8 (Fig. 7) which is one pH unit lower than the pH optimum of the stability of P: caudatum oxyHb. The spectrum of the final met H21 protein obtained after the autoxidation of the oxy form differs for reactions performed at alkaline or acidic pHs (Fig. 8). At pH 10, the final oxidation product obtained is the low-spin hexacoordinate form, while at pH 5, high-spin hexacoordinate Hb is obtained, a result very similar to that obtained for f! cuudatum Hb (Tsubamoto et al., 1990). At pH 5, the reaction proceeds via five isobestic points at 343, 412, 483, 526 and 596 nm, while at pH 10 the reaction proceeds via four isobestic points at 348, 486, 532 and 597 nm, with no intermediate form detected.

0. I 0.09

0.08 0.07

0.06

A

0.05 0.04 0.03

0.02 0.01

0

500 550 600 650 700 500 550 600 650 700 500 550 600 650 700

500 550 600 650 700

Wavelength (nm) Fig.6. Enzymic and non-enzymic reduction of metH2l Hb. (a) Incubation of the met H21 Hb in the ferredoxin/fetredoxin NADP' reductase The spectrum system. Spectra were recorded before (---) and after the addition of glucose-6-phosphate dehydrogenase (0 rnin and 3 min) (-). of the oxy form remained unchanged for at least 1 h. (b) Incubation of the met H21 Hb in the phenazine methosulfate/NADPH reducing system under a carbon monoxide atmosphere. The carbonmonoxy complex was formed within 30-40 min after the addition of NADPH and phenazine (c) Incubation of the oxyH21 protein in the phenazine methosulfatelNADPH reducing system. Spectra were recorded before methosulfate (-). (---) and after the addition of NADPH and phenazine methosulfate (0 min and 10 min) (-). (d) Incubation of horse heart metMb in the phenazine methosulfate/NADPH reducing system. Spectra were recorded before (- -) and after the addition of NADPH and phenazine methosulfate (0 min and 5 min) (-). All reactions were performed in 10 mM TridCI, pH 7 3 , at 23°C. Protein concentrations were 6.2 pM for H21 protein and 4 pM for horse heart niyoglobin. ~

Couture and Guertin (Eui: J. Biochem. 242)

A 0

s

0 0.25

r

N

I x

2

. -

0 5

-

150-

Table 1. Optical properties of Chfutraydomonusrecombinant Hbs. For pyridine hemochrome, the results are for the reduced minus oxidized spectrum. n.d., not determined. Derivative

0

5

I

7

----

a,

0.75

m

0 I

I

H19 Hb

50-

OXY

'

1.25 0

10

20

30

Maxima (absorbtion extinction coefficient) nm (mM-' cm-')

L

I, C

200

LI

I

-

B

785

5

6

7

8

9

1

0

PH

Time (hours)

Fig.7. The pH dependence of the autoxidation of the oxyH21 protein. (a) First-order plot for the autoxidation of the oxyH21 protein at pH 5-10 in 25 mM buffers at 23°C. The buffers used were sodium acetate, pH 5, MesfNaOH, pH 6, MopdNaOH, pH 7, Taps/NaOH, pH 8, Ches/NaOH, pH 9 and CapslNaOH, pH 10. (b) Plot of the calculated half-lives versus the pH.

deoxy carbonmonoxy met pH 5 met pH 7.5 cyanide azide dithiothreitol 2-mercaptoethanol pyridine hemochrome

412 (102), 545 (13), 581 (10) 426 (108), 529", 557 (15) 420 (142), 542 (11) 406 (130), 529 (6), 624 (2.6) 410 (IlO), 535 (8.5) 416 (97.6), 547 (11) 413 (IOS), 543 (9), 550" 423 (80.8), 543 (lo), 570" 423 (84), 547 (9.8) 570" 421, 524, 556

H20 Hb OXY

deoxy carbonmonoxy met, pH 5 met, pH 7.5 cyanide azide dithiothreitol 2-mercaptoethanol pyridine hemochrome

412, 545, 582 425, 529", 557 419,543 n. d. 410, 536 417, 547 416, 545, 580" 421,543, 572" 421, 545, 578" n. d.

H21 Hb OXY

475 525 515

625 675 415 525 575 625 C

Wavelength (nm) Fig. 8. Spectral changes with time for the autoxidation of the oxyH21 protein. Spectral changes during the autoxidation in 25 mM sodium acetate, pH 5 (left panel). These spectra are those recorded after 0, 1, 3 and 8.5 h. The final spectrum was recorded following the addition of potassium ferricyanide (- - -). (b) Spectral changes during the autoxidation in 25 mM CapsNaOH, pH 10 (right panel). These spectra are those recorded after 0, 5, 12 and 34 h. The final spectrum was recorded following the addition of potassium ferricyanide (- - -). The Hb concentration was 3.5 pM.

Properties of the H19 recombinant protein. The alignment of protozoan, Nostoc and Chlamydomonas Hb protein sequences shows that their amino termini are different (Fig. l a ) and that the H21 protein has an additional 14-19 amino acids at its amino terminus (Fig. 1a). To verify that these extra amino acids influence the properties of the recombinant Chlumydomonas Hb, a third protein (H19 Hb) was produced whose first amino acid following the initiating methionine (Ser44) corresponds to the first amino acid of I? caudatum Hb (Serl), the smaller protein of the group. The latter Hb has also high sequence similarity to Chlamydomonas Hbs at the amino terminus. As expected, the apparent molecular mass of the HI9 protein after SDSPAGE (1 2.5 kDa, Fig. 1d) is smaller than that of the native Chlamydomonus Hb (16 kDa; Fig. 1b). The H19 protein was also purified as a heme protein and the spectra of the oxy, met, deoxy and carbonmonoxy forms were determined to be nearly identical to those of the H20 and H21

deoxy carbon moiioxy met, pH 5 met, pH 7.5 cyanide azide dithiothreitol 2-mercaptoethanol pyridine hemochrome

412 (102), 545 (17), 581 (13) 423 (IOO), 529", 557 (14) 420 (141), 544 (12) 406 (130), 490 (7), 624 (4) 410 (110.3), 537 (10) 416 (97), 545 (11) 415 (103), 546 (9.6), 578" 422 (80.6), 544 (9.7), 572" n. d. 421, 524, 556

GST-H20 OXY

deoxy carbonmonoxy met, pH 7.5 *

412, 545, 581 425, 529", 557 419, 543 410, 536

Shoulder.

proteins (Table 1). Furthermore, the met H19 protein is reduced by NADPH and ferredoxidferredoxin NADP' reductase to give the oxy form, while the oxy form is converted to the met form in the presence of NADPH and phenazine methosulfate (result not shown). Finally, the half-life of the oxy complex measured at pH 8 (5 days) is very similar to that of the H21 protein. Therefore, at this level of analysis, the H19 protein behaved similarly to the H21 protein. Finally, characterization of the chimeric polypeptide resulting from the fusion of the GST protein (26 kDa) to the amino terminus of the H20 polypeptide (Couture et al., 1994), revealed that its spectra (oxy, deoxy, CO, met) are nearly identical to those of the H19, H20 and H21 proteins (Table 1) and that the oxy form is also reactive in the presence of phenazine methosulfate and NADPH (result not shown). The results obtained with the different recombinant proteins thus strongly suggest that the amino acids upstream of Ser44 are dispensable, at least under our experimental conditions. Therefore, although the amino-terminal sequence of the mature L1637

786

Couture and Guertin ( E m J. Biochern. 242)

protein is still unknown, we may expect that it should have similar if not identical properties to the recombinant proteins.

DISCUSSION The evidence presented here indicates that the chloroplast LI637 polypeptide of the green unicellular alga C. eugumetos is a heme protein that reversibly binds dioxygen. Due to the low levels of this protein in C. eugametos, a recombinant version of this protein was expressed and characterized. An important consideration in this approach is whether the recombinant heme protein accurately reflects the wild-type version, particularly as the amino terminus of the latter has not been unequivocably established. The electrophoretic mobility of the recombinant proteins H20 and H21 indicates that their amino termini are within four or five amino acids of the wild-type protein (Fig. 1b and d). Significantly, recombinant proteins whose amino termini are shorter (H19) or longer (GST-H20) have similar spectra and reactivities as H21 (Table 1). These findings indicate that, even if H21 differs from the wild-type protein by few amino acids at its amino terminus, these residues do not significantly influence the biochemical properties of the protein, and that at the current level of analysis, the properties of the recombinant protein reflect those of the wild-type protein. The ultraviolethisible spectrum of the oxy form of recombinant Chlamydomonas Hb is very similar to those of protozoan Hbs and of the cyanoglobin. The most distinctive feature of these spectra is the intensity of the (I peak, which is always less peak ratio values determined than that of the p peak. The (I/P~,.,~ for the Chlamydomonas recombinant proteins (0.76) falls in the range observed for the cyanoglobin (0.6) and Z? cuudafum Hb (0.94). This characteristic is not unique to the latter group of Hbs as it has also been observed for FHbs (Ioannidis et al., 1992; Probst et a]., 1979), however it contrasts to the situation observed in vertebrate Hbs and Mbs. In contrast to the met form of Hbs and Mbs, which usually contains a ferric iron in a high-spin state at neutral pH, the met form of C. eugarnetos Hbs is predominantly composed of a lowspin hexacoordinate heme (putative hemichrome) at neutral pH while, at acidic pH, a high-spin hexacoordinate heme is present. This is similar to the situation encountered for the met cyanoglobin but contrasls the situation observed for f! caudatum and 7: pyrvimnis Hbs whose met form contain a high-spin hexacoordinate heme at neutral pH (Iwaasa et al., 1990; Tsubamoto et al., 1990). However, Z? caudatum Hb has been showed to form a hemichrome upon prolonged storage of the met form at neutral pH or during the autoxidation of the oxy form at alkaline pH (Tsubamoto et al., 1990). Although the met form of Chlamydomonas Hb and cyanoglobin are spectroscopically similar, they react differently with anionic ligands. Cyanoglobin binds cyanide only at acidic pH, i.e. when the high-spin hexacoordinate heme is present (Thorsteinsson et al., 1996) while the C. eugamefos Hb binds both azide and cyanide in the high-spin and low-spin hexacoordinate heme forms. It seems, therefore, that the coordination between the heme and the distal ligand of the hemichrome is more easily disrupted in C. eugametos Hb than in the cyanoglobin. In addition to cyanide and azide, the low-spin hexacoordinate heme of C. eugametos Hb also binds the sulfurous compounds 2-mercaptoethanol and dithiothreitol but not the weak field ligand fluoride. The distal amino acid ligand of the putative Chlamydomonas hemichrome is unknown and it could occur somewhere in the sequence other than helix E7. Alignment of the sequences of C. eugametos, protozoa and Nostoc Hbs with the sequences of Hbs

of known three-dimensional structure indicates that the amino acid present on the distal side is glutamine instead of a histidine residue (Fig. 4; Couture et al., 1994) while His1 11 at position (F8) is the proximal ligand. However, Chkzmydonzonas Hbs, as well as those of Paramecium, Tetmhymena and Nostoc, have one or two other histidine residues that could be involved i n the coordination of the heme iron atom. Further investigation is thus necessary to identify the distal ligand. The reaction of the recombinant Ch1ainydomona.s Hbs with reducing compounds in the presence of electron carriers merits discussion. When NADPH is used in combination with ferredoxin and ferredoxin NADP' reductase, the met form of Chlumydomonas Hb is readily converted to the oxy form indicating an efficient electron transfer from NADPH to the heme. Phenazine methosulfate also mediates the transfer of the electron necessary to reduce the ferric iron atom of heme because the C O complex is formed when the reaction is performed under a C O atmosphere. However, the bound dioxygen molecule of the oxy form is reactive in the presence of NADPH and phenazine methosulfate. This phenomenon is observed when the oxy form is incubated in the presence of NADPH and phenazine methosulfate, which causes the rapid conversion of the oxy form to the met form (Fig. 6c). The product generated by the reduction of the dioxygen molecule is unknown but could be hydrogen peroxide if one electron is provided by the iron atom and the other by the NADPH/phenazine methosulfate couple. The reduction of the dioxygen molecule in the presence of reducing conipounds has been previously observed for human oxyHb which generated, in the presence of the small compound aquopentacyanoferrate(II), peroxide and metHb but was stable in the presence of the bulkier Fe(1I)EDTA molecule (Kawanishi and Caughey, 3 98s). In this case, a steric hindrance was postulated as the major factor governing the reactivity of the bound dioxygen. The physiological relevance of the reaction of Chlumydomonas Hbs with phenazine methosulfate is unknown but it indicates the possibility that the heme pocket of the LI637 protein is more accessible to small molecules than the heme pocket of horse heart myoglobin. In such a case, steric hindrance could explain the reactivity of the dioxygen molecule in the presence of phenazine methosulfate but not of ferredoxin/ferredoxin NADP' reductase. A similar reaction could also be responsible for the failure of the reduction procedure employing sodium dithionite in the presence of oxygen. It should be noted that ferredoxin is a normal constituant of the photosynthetic electron-transport chain i n the chloroplast of plants and green algae and could, therefore, be responsible for the maintenance of Chlarnydonioriu.~Hb in the reduced state in v i v a The current results demonstrate that recombinant Chlamydomorzas Hbs are relatively resistant to autoxidation, having a halflife of 7 days at pH 8, the pH found in the chloroplast under illumination. Chlamydomonas Hb thus appears to be more resistant to autoxidation than Paramecium and Nostoc Hbs, probably as a result of a different evolutionary pressure imposed by the environment. The requirement for a slow autoxidizing protein is probably critical in view of the oxygen-rich (and more oxidizing) environment of the chloroplast as opposed to oxygen-poor environments where the evolutionary pressure for slow autoxidizing hemoproteins may not be as high. In oxygen-poor environments, faster autoxidizing proteins are found such as the oxygen sensor of Rhizobium meliloti, a bacteria living in plant nodules (half-life of 15-20 min) and cynaoglobin which is produced only in cells grown under hypoxic condition (half-life of less than 1 2 h). The biological function of Chkarnydomonas Hb remains unknown. Oxygen sensing seems unlikely in view of the high oxygen content of the chloroplast. These proteins could serve to

Couture and Guertin ( E m J. Biochem. 242) protect oxygen-sensitive enzymes, perhaps as part of macromolecular complexes. Alternatively, they could function in the transport of oxygen to the terminal oxidase involved in chlororespiration, a reaction thought to recycle the NADH produced by the catabolism of starch during the dark period. We thank Lindsay Eltis for access to the anaerobic chamber and helpful comments. This work was supported by the Natural Sciences and Engineering Research Council of Canada grant 06P0046306 and by the Fonds pour la Formation de Chercheurs et l’aide u la Recherche grant 96ER0350 to M. G. M. C. is a recipient of a graduate scholarship from the Fonds pour la Formation de Chercheurs er 1 ’aide 2 la Recherche.

REFERENCES Appleby, C. A. (1978) Purification of Rhizobium cytochromes P-450, Methods Enzymol. 52, 157- 166. Bashford, D., Chothia, C. & Lesk, A. M. (1987) Determinants of a protein fold. Unique features of the globin anlino acid sequences, J. Mol. Biol. 196, 199-216. Beck von Bodman, S., Schuler, M. A,, Jollie, D. R. & Sligar, S. G. (1986) Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5, Proc. Nut1 Acad. Sci. USA 83, 9443-9447. Couture, M.. Chamberland, H., St-Pierre, B., Lafontaine, J. & Guertin, M. (1994) Nuclear genes encoding chloroplast hernoglobins in the unicellular green alga Chlamydomonas eugametos, Mol. Gen. Genet. 243, 185-197. Cramni, R., Siddiqui, R. A. & Friedrich, B. (1994) Primary sequence and evidence for a physiological function of the flavohemoprotein of Alcaligenes eutrophus, J. Bid. Chem. 269, 7349-7354. Darawshe, S., Tsafadyah, Y. & Daniel, E. (1987) Quaternary structure of erythrocruorin from the nematode Ascaris suum. Evidence for unsaturated haem-binding sites, Biochem. J. 242, 689-694. Deng, W. P. & Nickoloff, J. A. (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site, Anal. Biochem. 200, 81 -88. Ermler, U., Siddiqui, R. A,, Cramm, R. & Friedrich, B. (1995) Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution, EMBO J. 14, 6067-6077. Favey, S., Labesse, G., Vouille, V. & Boccara, M. (1995) Flavohaemoglobin HmpX: a new pathogenicity determinant in Erwinia chrysanthemi strain 3937, Microbiol. 141, 863-871. Francis, R. T. & Becker, R. R. (1984) Specific indication of hemoproteins in polyacrylamide gels using a double-staining process, Anal. Biochem. 136, 509-514. Gagne, G. & Guertin, M. (1992) The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under lightidark cycles involves genes that represent direct responses to light and photosynthesis, Plant Mol. Bid. 18, 429-445. Hayashi, A,, Suzulu, T. & Shin, M. (1973) An enzymic reduction system for metmyoglobin and methemoglobin, and its application to functional studies of oxygen carriers, Biochim. Biophys. Acta 310, 309316. Ioannidis, N., Cooper, C. E. & Poole, R. K. (1992) Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli, Biochem. J. 288, 649-655. IWadSa, H., Takagi, T. & Shikama, K. (1989) Protozoan myoglobin from Paramecium caudatum. Its unusual amino acid sequence, J. Mol. B i d . 208, 355-358. Iwaasa, H., Takagi, T. & Shikama, K. (1990) Protozoan hemoglobin from Tetrahymena pyriformis. Isolation, characterization, and amino acid sequence, J. Bid. Chem. 265, 8603-8609.

787

Iwaasa, H., Takagi, T. & Shikama, K. (1992) Amino acid sequence of yeast hemoglobin. A two-domain structure, J. Mol. Bid. 227, 948954. Kajita, A., Noguchi, K. & Shukuya, R. (1970) A simple non-enzymatic method to regenerate oxyhemoglobin from methemoglobin, Biochem. Biophys. Res. Commun. 39, 1199-1204. Kawanishi, S. & Caughey, W. S. (1985) Mechanism of electron transfer to coordinated dioxygen of oxyheinoglobins to yield peroxide and methemoglobin. Protein control of electron donation by aquopentacyanoferrate(II), J. Biol. Chem. 260, 4622-4631. Keegstra, K. & Olsen, L. J. (1989) Chloroplastic precursors and their transport across the envelope membrane, Annu. Rev. Planr Physiol. Plant Mol. Biol. 40, 471 -501. Moens, L., Vanfleteren, J., Van de Peer, Y., Peeters, K., Kapp, 0. Czeluzniak, J., Goodman, M., Blaxter, M. & Vinogradov, S. (1996) Globins in nonvertebrate species - dispersal by horizontal gene transfer and evolution of the structure-function-relationships, Mol. Biol.E w ~ .13, 324-333. Pathirana, S. M. & Tjepkema, J. D. (1995) Purification of hemoglobin from the actinorhizal root-nodules of Myrica-gale I, Plant Physiol. 0107, 827-831. Poole, R. K., Baines, B. S. & Appleby, C. (1986) Haemoprotein b-590 (Escherichia coli), a reducible catalase and peroxidase: Evidence for its close relationship to hydroperoxidase I and a ‘cytochrome a,b’ preparation, J . Gen. Microbiol. 132, 1525-1539. Potts, M., Angeloni, S. V., Ebel, R. E. & Bassam, D. (1992) Myoglobin in a cyanobacterium, Science 256, 1690-1692. Probst, I., Wolf, G. & Schlegel, H. G. (1979) An oxygen-binding flavohemoprotein from Alcaligenes eutrophus, Biochim. Biophys. Acta 576, 471 -478. Smith, M. H., George, P. & Preer, J. R. J. (1962) Preliminary observations on isolated Purumecium hemoglobin, Arch. Biochem. Biophys. 99, 313-318. Takagi, T., Iwaasa, H., Yuasa, H., Shikama, K., Takemasa, T. & Watanabe, Y. (1993) Primary structure of Tetrahymena hemoglobins, Biochim. Biophys. Acta 1173, 75-78. Thorsteinsson, M. V., Bevan, D. R., Ebel, R. E., Weber, R. E. & Potts, M. (1996) Spectroscopical and functional characterization of the hemoglobin of Nostoc-commune UTEX-584 (cyanobacteria), Biochim. Biophys. Acra-Prot. Struct. 1292, 133-139. Tsubamoto, Y., Matsuoka, A,, Yusa, K. & Shikama, K. (1990) Protozoan myoglobin from Paramecium caudatum. Its autoxidation reaction and hemichrome formation, EUK J. Biochem. 193, 55-59. Usuki, I. & Hino, A. (1991) Changes in relative concentrations of the hemoglobin components in Paramecium caused by cell growth and temperature of the culture, 1. Cell. Sci. 100, 635-639. Vasudevan, S. G., Armarego, W. L., Shaw, D. C., Lilley, P. E., Dixon, N. E. & Poole, R. K. (1991) Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12, Mol. Gen. Genet. 226, 49-58. Wakabayashi, S., Matsubara, H. & Webster, D. A. (1986) Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla, Nurure 322,481 -483. Wood, P. M. (1984) Bacterial proteins with CO-binding b- or c-type haem. Functions and absorption spectroscopy, Biochim. Biophys. Acta 768, 293-317. Yamauchi, K., Tada, H. & Usuki, I. (1995) Structure and evolution of Paramecium hemoglobin genes, Biochim. Biophys. Acta 1264, 53 62. Zhu, H. & Riggs, A. F. (1992) Yeast flavohemoglobin is an ancient protein related to globins and a reductase family, Proc. Nut/ Acad. Sci. USA 89, 5015-5019.