JOURNAL OF BACrERIOLOGY, Nov. 1994, p. 6663-6671 Vol. 176, No. 21 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology Bacillus subtilis CtaA Is a -Containing Membrane Protein Involved in Biosynthesis BIRGITFA SVENSSON AND LARS HEDERSTEDT* Department ofMicrobiology, Lund University, S-22362 Lund, Sweden Received 27 May 1994/Accepted 29 August 1994 Heme A is a of many respiratory oxidases. It is synthesized from protoheme IX (heme B) seemingly with heme 0 as a stable intermediate. The Bacilus subtilis ctaA and ctaB genes are required for heme A and heme 0 synthesis, respectively (B. Svensson, M. Lubben, and L. Hederstedt, Mol. Microbiol. 10:193-201, 1993). Tentatively, CtaA is involved in the monooxygenation and oxidation of the methyl side group on ring D in heme A synthesis from heme B. B. subtilis ctaA and ctaB on plasmids in both B. subtilis and Escherichia coil were found to result in a novel membrane-bound heme-containing protein with the characteristics of a low-spin b-type cytochrome. It can be reduced via the respiratory chain, and in the reduced state it shows light absorption maxima at 428, 528, and 558 nm and the a-band is split. Purified cytochrome isolated from both B. subtilis and E. coli membranes contained one polypeptide identified as CtaA by sequence analysis, about 0.2 mol of heme B per mol of polypeptide, and small amounts of heme A.

The gram-positive aerobic soil bacterium Bacillus subtilis can O from farnesyl PP1 and protoheme IX (29, 30). The properties synthesize a-, b-, c-, d-, and o-type cytochromes (41), which of B. subtilis ctaB mutants are consistent with this function of contain heme A, protoheme IX (heme B), , heme D, CtaB in heme 0 synthesis (37). and heme 0, respectively. Four different terminal oxidases B. subtilis CtaA is a 34-kDa integral membrane protein, as have been found in B. subtilis: two aa3-type cytochromes (the predicted from the DNA sequence (22). It is not required for aa3 and the caa3 oxidases), a cytochrome d, and a cytochrome heme 0 synthesis (37). Our recent data from studies of B. o. The cytochrome aa3 is a menaquinol oxidase, and the subtilis and E. coli strongly suggest that CtaA is involved in the cytochrome caa3 is a cytochrome c oxidase (17, 33, 34). monooxygenation and oxidation of the methyl side group on Cytochrome o has been identified by photo- porphyrin ring D required to make heme A (37). This modi- dissociation spectra (16) and by the demonstration of heme 0 fication step is analogous to the biotransformation of chloro- in particular mutants lacking cytochrome a (37). Cytochrome d phyll a into b, in which a methyl group on has been identified only spectroscopically and by the presence ring B is modified into a formyl group (26, 36). Chlorophyll a of heme D in membranes (1). and b are prosthetic groups of the light-harvesting complex in Heme 0 differs structurally from protoheme IX in that there green plants and algae. The catalyzing these methyl- is a hydroxyethyl farnesyl side chain on porphyrin ring A to-formyl modification reactions have not been identified for instead of a vinyl group (42). Heme A further differs from any organism. heme 0 in that there is a formyl side group on porphyrin ring To understand better the role of B. subtilis CtaA and CtaB D instead of a methyl group. Heme A is synthesized from in heme A synthesis, we have now analyzed membranes from protoheme IX, probably with heme 0 as a stable intermediate B. subtilis and E. coli strains containing ctaA and ctaB on (for structures of heme 0 and heme A and the tentative heme plasmids. A membrane-bound heme-protein that had not been A biosynthetic pathway, see, e.g., Fig. 1 of reference 37). Very previously described, with the characteristics of a cytochrome little is known about enzymes for heme A biosynthesis in b, was found to result from these genes. The cytochrome eukaryotic cells and bacteria. polypeptide was isolated and identified as the ctaA gene In B. subtilis, we have identified two genes required for heme product. A biosynthesis, ctaA and ctaB (37). These two adjacent genes map at 127 degrees on the chromosomal map, they are divergently transcribed from overlapping promoters, and they MATERIALS AND METHODS are located immediately upstream of the ctaCDEF gene cluster encoding subunits II, I, III, and IVB, respectively, of the Bacterial strains and plasmids. B. subtilis strains used were cytochrome caa3 (34, 39). ctaA genes have so far been found 3G18 (trpC2 ade met) (obtained from G. Venema, University only in Bacillus species (21, 22, 27). Genes corresponding to B. of Groningen, Groningen, The Netherlands), KA44109 (sdhC subtilis ctaB have been found in Saccharomyces cerevisiae 109 ilvB2 aecA5) (25), and 3G18sdhAl12 (trpC2 ade met (COX10), in other Bacillus species, and in gram-negative AsdhCA::cat) (7). The E. coli strain used was JM109 [recAl bacteria, e.g., Paracoccus denitrificans (ctaB) and Escherichia endAl gyrA96 thi hsdR17 supE44 reL1 X- A(lac-proAB) (F' coli (cyoE) (4, 24, 27, 28). CyoE is an integral membrane traD36 proAB lacIZAM15)] (44). Plasmids used in this work protein (3) which recently has been identified as heme 0 are presented in Table 1. synthetase, i.e., an that catalyzes the synthesis of heme Growth of bacteria and preparation of membranes. B. subtilis strains were kept on tryptose blood agar base plates (Difco Laboratories) supplemented with 5 g of glucose liter-1. * Corresponding author. Mailing address: Department of Microbi- E. coli was kept on Luria-Bertani (LB) agar plates. Antibiotics ology, Lund University, Solvegatan 21, S-22362 Lund, Sweden. Phone: were added to media at the following final concentrations: 46-46-108622. Fax: 46 46 157839. ampicillin, 35 mg (plates) and 50 mg (liquid cultures) liter-'; 6663 6664 SVENSSON AND HEDERSTEDT J. BACTERIOL.

TABLE 1. Plasmids used in this work Centricon-30 concentrators (Amicon). The concentrated sam- ple was applied on a Sephacryl S-300 (Pharmacia) column (2.6 Plasmid Descriptiona Reference by 90 cm). The column was eluted with 20 mM MOPS buffer pUC19 Apr 44 with 0.1% Thesit at a flow rate of 30 mi/h, and 5-ml fractions pHP13 Emr Cmr; E. coli-B. subtilis 10 were collected. Peak fractions were pooled as shown in Fig. 3. shuttle vector Different final purification steps were employed depending pAI536 pycA'ctaABC Apr Cmr 21 pCTA1301 ctaB in pHP13 This work on whether the cytochrome was isolated from B. subtilis pCTA1302 ctaA in pHP13 This work 3G18sdhA12/pCTA1303 membranes or from E. coli JM109/ pCTA1303 ctaA ctaB in pHP13 This work pCfA1900 membranes. The Sephacryl S-300 pool obtained pCTA1900 ctaA ctaB in pUC19 37 from B. subtilis was loaded at a flow rate of 1 ml/min on a pCTA1901 ctaA in pUC19 37 hydroxylapatite minicolumn (HTP-cartridge; Bio-Rad) equili- pCTA1903 ctaB in pUC19 37 brated in 10 mM Na phosphate in 20 mM Na MOPS/HCl pCTA177B ctaB in pACYC177; Kmr 37 buffer, pH 7.4, containing 0.1% Thesit. The flowthrough a Apr, Cmr, Emr and Kmr indicate that the plasmid confers ampicillin, fraction, which contained cytochrome b-CTA, was concen- chloramphenicol, erythromycin and kanamycin resistance, respectively. trated using a Centricon-30 concentrator. The Sephacryl S-300 pool obtained from E. coli JM109/pCTA1900 was supple- mented with Na phosphate to a final concentration of 10 mM and then loaded on the hydroxylapatite minicolumn at a flow chloramphenicol, 5 mg (B. subtilis) and 20 mg (E. coli) liter-'; 1 The cartridge was washed with 10 ml of 10 erythromycin, 5 mg liter-'; and kanamycin, 10 mg liter-'. rate of ml/min. Bacteria were grown in 1-liter portions in 5-liter indented E mM Na phosphate in 20 mM MOPS buffer (pH 7.4)-0.1% flasks at 37°C on a rotary shaker at 200 rpm and harvested Thesit. Bound material was eluted using a two-step profile and when the cultures reached early stationary phase. B. subtilis then by a 20-ml gradient, 300 to 500 mM Na phosphate in 20 membranes were prepared from cells grown in nutrient sporu- mM Na MOPS/HCl (pH 7.4)-0.1% Thesit (see Fig. 3). The lation medium phosphate (NSMP) supplemented with 5 g of gradient was run at a flow rate of 0.5 mlmin, and 2-ml fractions glucose or 5 g of glycerol liter1- (12). E. coli membranes were were collected. Peak fractions were pooled as shown in Fig. 3 prepared from cells grown in LB medium as described by and were concentrated as described above. Friden et al. (6). Membrane preparations were stored in 20 During the development of the purification procedures, we mM sodium MOPS (3[N-morpholino]propanesulfonate)/HCl noted that in the presence of Mg2e ions (mM concentrations), buffer (pH 7.4) at -70°C. cytochrome b-CTA from E. coli extracts could not be eluted Construction of plasmids and transformation of strains. from the DEAE-Sephacel as a distinct peak. Furthermore, the General DNA techniques were as described in Sambrook et al. behavior of the cytochrome isolated from E. coli was variable (32). For the overexpression of ctaA and ctaB in B. subtilis, the on the hydroxylapatite minicolumn. Some preparations eluted 2.7-kb BglII-SpeI fragment of pCIA1900 was cloned in pHP13, at 200 mM; others eluted at up to 500 mM Na phosphate. We giving plasmid pCTA1303. Plasmid pCTA1301 was con- do not understand the reason for this variability. structed by cloning a 2-kb BglII-SpeI fragment, containing SDS-PAGE and N-terminal amino acid analysis. Sodium ctaB, from pAI536 into pHP13. Plasmid pCTA1302 was ob- dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) tained by cloning a 1.6-kb fragment, containing ctaA, from was carried out according to the method described by Schagger pCTA1901 into pHP13. Maps of plasmids pCTA1900 and and von Jagow (35). Samples were routinely denatured in the pCTA1901 have recently been published elsewhere (37). presence of 2% (wt/vol) SDS at 40°C for 30 min just before Competent B. subtilis and E. coli cells were prepared as electrophoresis. described by Hoch (14) and by Mandel and Higa (19), respec- For N-terminal sequence analysis of the cytochrome b-CTA tively. polypeptide, it was first run in SDS-PAGE (10% acrylamide) Cytochrome b-CTA purification procedure. Membranes and then electroblotted to a polyvinylidene difluoride (PVDF) from B. subtilis 3G18sdhA12/pCTA1303 or E. coli JM109/ membrane (Immobilon P filter; Millipore) using a semidry pCTA1900 at 10 mg of protein per ml were solubilized with 4% blotting apparatus (Semi-Dry Electroblotter B; ANCOS). A 10 (wt/vol) Thesit (nonionic detergent; dodecyl poly[ethylene mM concentration of 3-cyclohexanylamino-1-propanesulfonic glycol ether]) (Boehringer Mannheim) in 20 mM Na MOPS/ acid (CAPS), pH 11.0, in 10% (vol/vol) methanol was used as HCl buffer, pH 7.4, in the presence of 0.3 mM phenylmethyl- electrotransfer buffer. After blotting (250 mA, 75 min), the sulfonyl fluoride. The solubilization mixture was incubated for membrane was washed in transfer buffer (twice for 20 min), 15 min at room temperature and then centrifuged at 20,000 stained with 0.1% Coomassie blue R250 in 50% methanol for rpm (Beckman JA20 rotor) for 45 min at 4°C. The pellet was 1 to 2 min, and destained in 50% methanol-10% acetic acid for discarded, and the supernatant was centrifuged at 40,000 rpm 5 min. The membrane was finally washed several times in (Beckman Ti 50.2 rotor) for 45 min at 4°C. The final superna- high-performance liquid chromatography (HPLC)-quality wa- tant was diluted with 1 volume of 20 mM Na MOPS/HCl, pH ter, dried in air, and stored dry at -20°C until used. To obtain 7.4, and then applied to a 50-ml DEAE-Sephacel (Pharmacia) internal amino acid sequence data from cytochrome b-CTA, column (radius, 13 mm) at a flow rate of 20 mi/h. The column the purified protein (2 nmol of protoheme IX) was treated with was washed with 2 bed volumes of MOPS buffer containing trypsin (pretreated with N-tosyl-1-phenylalanine chloromethyl 0.1% (wtlvol) Thesit. Bound proteins were eluted with 300 ml ketone [Worthington]) at 100:1 (wt/wt) in 20 mM Na MOPS/ of a 0 to 600 mM NaCl gradient in 20 mM Na MOPS/HCl (pH HCl buffer (pH 7.4)--l1% (wt/vol) Thesit at 37°C for 2 h. 7.4)-0.1% (wtlvol) Thesit. Fractions of 3 ml were collected and Phenylnethylsulfonyl fluoride was then added to a final con- analyzed for light absorption at 280 nm (protein) and 410 or centration of 0.3 mM before the sample was denatured by 414 nm (cytochrome). Fractions were pooled as shown in Fig. incubation in the presence of 2% (wt/vol) SDS and loaded on 3 and dialyzed overnight at 4°C against 20 mM MOPS buffer an SDS-14% polyacrylamide gel. After SDS-PAGE, the and concentrated about fivefold using solid polyethylene glycol polypeptides were electroblotted to a PVDF membrane and 20,000 (Merck) and dialysis tubing (cutoff, 6 to 8 kDa) or using stained as described above. Edman degradation of blotted VOL. 176, 1994 HEME A BIOSYNTHESIS IN BACILLUS SUBTILIS 6665

A B C D

a) C) e m ~~~~o 0CO) 6

500 550 600 650 700 500 550 600 650 700 500 550 6BO 650 700 Wavelength (nm) FIG. 1. Light absorption difference (dithionite reduced minus oxidized) spectra at 77 K of membranes isolated from B. subtilis 3G18/pHP13 (A), 3G18/pCTA1303 (B), KA44109/pHP13 (C), and KA44109/pCTA1303 (D). 3G18 is the wild-type strain, and KA44109 is defective in (SdhC). Plasmid pCTA1303 is a derivative of pHP13 carrying B. subtilis ctaA and ctaB. The protein concentration was 10 mg/ml, and the scan speed was 50 nm/min. The fourth derivative of the absorption spectrum is shown at the top in each panel. The vertical bar indicates the absorbance scale.

polypeptides was performed at the University of Agriculture, has a split a-band absorption peak at about 556 nm and a Uppsala, Sweden. ,3-peak at 528 nm at 77 K. The absorption maxima of the split Other methods. Light absorption spectroscopy was done peak were found at 553 and 559 nm, as shown by the fourth using a Shimadzu UV3000 spectrophotometer for low-temper- derivative of the spectrum (Fig. 1B, top). The absorption peaks ature spectra and a Shimadzu UV2101-PC spectrophotometer seen at about 600 nm and at about 625 nm in the spectra shown for room temperature spectra. A 1-nm slit was used on both in Fig. 1 are from cytochrome a and cytochrome d, respec- machines. Cuvettes with light paths of 10 and 4 mm were used tively, whereas the peak at about 560 nm results from b- and for room temperature and 77 K spectroscopy, respectively. c-type cytochromes (41). The presence of ctaA and ctaB on Membranes were reduced with 10 mM K succinate, 10 mM plasmids did not affect the concentrations of cytochrome a and glycerol-3-phosphate, 10 mM ascorbate, 0.1 mM NNN',N'- cytochrome d in membranes. tetramethyl-p-phenylene-diamine hydrochloride (TMPD), or The low-temperature light absorption spectrum of the chro- with a few grains of solid sodium dithionite and oxidized with mophore in 3G18/pCTA1303 membranes (Fig. 1B) is very 1 mM K3Fe(CN)6. HPLC analysis of proteins was done using similar to that of cytochrome b-558 of the B. subtilis succinate- a Beckman SEC 4000 (7.5 mm by 30 cm) gel filtration column menaquinone reductase (succinate dehydrogenase) which in and a Waters 991 photodiode array detector. The flow rate was the dithionite reduced state shows a split a-band absorption 0.7 ml/min, and the buffer used was 20 mM Na MOPS/HC1 peak with maxima at 553 and 558 nm at 77 K (9). Cytochrome (pH 7.4)-0.1% (wt/vol) Thesit. Heme was analyzed by re- b-558 dominates the 560-nm region in spectra of wild-type versed-phase HPLC as described before (37) and by the membranes (9) (Fig. 1). To eliminate cytochrome b-558 and pyridine hemochromogen spectrum (5). Protein concentra- make the novel chromophore more clearly visible, we made tions were determined according to the method described by use of B. subtilis KA44109 which has a nonsense mutation Lowry et al. (18) with bovine serum albumin as the standard. (sdhClO9) in the middle of the sdhC gene (8, 25). Membranes Quantitative and total amino acid analyses on samples from KA44109/pCTA1303 contained the chromophore corre- hydrolyzed in 6 N HC1 for 24 and 72 h were done at the Central lated with ctaAB, which demonstrates that it is unrelated to the Amino Acid Laboratory, Uppsala, Sweden. Iron and copper cytochrome b-558 of the succinate-menaquinone reductase amounts were determined by atomic absorption at Analytica (Fig. 1C and D). This finding was corroborated by difference AB, Stockholm, Sweden. As a control for the background spectra of membranes from B. subtilis 3G18sdhAl12 containing (free) metal content, a part of the cytochrome b-CTA prepa- pCTA1303 which were similar to those of KA44109/pCTA ration was concentrated using a Centricon-30 concentrator, 1303. Strain 3G18sdhA12 has the sdhC gene deleted from the and the metal content in the effluent (buffer) was also ana- chromosome (7). lyzed. The novel chromophore resulting from ctaA and ctaB on plasmids will be called cytochrome b-CTA in the following to RESULTS discriminate it from the diheme cytochrome b-558 of the succinate-menaquinone reductase. A novel B. subtilis cytochrome b. A chromophore with the B. subtilis cytochrome b-CTA in E. coli. E. coli cells do not spectral features of a cytochrome was detected in dithionite- normally contain heme A, but heme A is synthesized and reduced membranes isolated from the wild-type B. subtilis accumulates in the membrane if the B. subtilis ctaA gene is strain 3G18 containing plasmid pCTA1303 (Fig. 1B). This present on plasmids (37). Larger amounts of heme A are found plasmid is a derivative of pHP13, with a copy number of about in E. coli membranes when both ctaA and ctaB are present. five (10), containing the B. subtilis ctaA and ctaB genes under The ctaB gene alone on plasmids in E. coli does not result in their native promoters (Table 1). Membranes from B. subtilis detectable amounts of heme A in membranes. 3G18 carrying plasmid pCTA1302 or pCTA1301, i.e., pHP13 Cytochrome b-CTA was found in membranes from E. coli with ctaA or ctaB, respectively, did not display the chro- JM109 containing pCTA1900 which carries B. subtilis ctaA and mophore (spectra not shown) and showed spectra similar to ctaB and is a derivative of the high-copy-number vector pUC19 those of membranes from 3G18/pHP13 (Fig. 1A). The differ- (Fig. 2B). Plasmids carrying either the ctaA or the ctaB gene ence (reduced minus oxidized) spectrum of the chromophore did not result in detectable amounts of cytochrome b-CTA in 6666 SVENSSON AND HEDERSTEDT J. BACT1ERIOL. A B BC D E

L I -V -2 0 CO) .0

500 550 6o0 500 550 600 500 550 6oo 500 550 6oo

Wavelength (nm) FIG. 2. Light absorption difference (dithionite reduced minus oxidized) spectra at 77 K of membranes isolated from E. coli JM109 containing different plasmids as follows: pUC19 (A), pCTA1900 (ctaAB) (B), pCTA1901 (ctaA) (C), pCTA1903 (ctaB) (D), and pCTA1901 (ctaA) plus pCTA177B (ctaB) (E). The protein concentration was 15 mg/ml, and the scan speed was 50 nm/min. The fourth derivative of the absorption spectrum is shown at the top in each panel. The vertical bar indicates the absorbance scale.

E. coli JM109 (Fig. 2C and D). These results obtained in E. coli b-CTA in E. coli is correlated with the synthesis and accumu- were consistent with our findings in B. subtilis. To exclude the lation of significant amounts of heme A in membranes. possibility that ctaA or ctaB is not expressed from pCTA1901 Purification of B. subtilis cytochrome b-CTA. Membranes (ctaA) or pCTA1903 (ctaB), two experiments were done. First, isolated from B. subtilis 3G18sdhA12/pCTA1303 and E. coli the ctaB gene of pCTA1903 was cloned into pCTA1901, JM109/pCTA1900 were used as starting materials to isolate leading to the reconstruction of the ctaAB region. As expected, cytochrome b-CTA. The cytochrome was solubilized from the reconstructed plasmid directed production of cytochrome membranes by using the nonionic detergent Thesit and was b-CTA (spectrum not shown). Second, E. coli JM109/ purified by using DEAE-Sephacel, Sephacryl S-300, and hy- pCTA1901 was transformed with pCTA177B, which is a deriv- droxylapatite chromatography in the presence of 0.1% (wt/vol) ative of pACYC177 containing ctaB on a fragment correspond- Thesit as described in Materials and Methods. Chromatogra- ing to that in pCTA1903. Membranes of JM109/pCTA1901/ phy profiles are shown in Fig. 3, and quantitative aspects of pCTA177B contained cytochrome b-CIA (Fig. 2E), which representative purifications are presented in Table 3. Cyto- demonstrates that these genes can also function in trans. The chrome b-CTA from B. subtilis 3G18sdhA12/pCTA1303 and E. results, summarized in Table 2, show that (over)production of coli JM109/pCTA1900 membranes, respectively, fractionated cytochrome b-CTA requires both ctaA and ctaB and strongly identically on the DEAE-Sephacel and Sephacryl S-300 col- suggest that one or both of these genes is the structural gene umns but differently on the hydroxylapatite column; the cyto- for this cytochrome. Furthermore, the presence of cytochrome chrome extracted from B. subtilis did not bind to hydroxylapa- tite, whereas that from E. coli was bound. SDS-PAGE of the purified cytochromes from both sources showed a band with an mass of 23 TABLE 2. Production of B. subtilis cytochrome b-CIA from single polypeptide apparent plasmids in E. coli JM109 kDa (Fig. 4). This polypeptide was absent from membranes of the negative control E. coli JM109/pUC19 (gels not shown). cta gene(s) The CtaA and CtaB polypeptides have masses of 34.1 and 33.8 Plasmid(s) present Cytochrome b-CTA Heme A in in membranesa membranesb kDa, respectively, as deduced from the DNA sequences (21, ctaA ctaB 34). Both polypeptides are predicted to be very hydrophobic pUC19 - - - - and may migrate aberrantly on SDS-polyacrylamide gels, i.e., pCTA1900 + + + + could appear as 23-kDa polypeptides. The CtaB homolog pCTA1901 + - _ (+)C CyoE, which has a predicted mass of 32 kDa, migrates on pCTA1903 - + - - SDS-polyacrylamide gels as a 26- to 28-kDa polypeptide (29). pCTA1901 + pCTA177B + + + + The isolated cytochrome b-CI7A protein showed another prop- (trans) erty in common with some very hydrophobic proteins, such as a Determined by light absorption spectroscopy. subunit I of cytochrome oxidases. It aggregated and remained b Determined by reversed-phase HPLC. at the top of the gel during SDS-PAGE when boiled in the c Only low amounts of heme A are found (37). presence of 2% (wt/vol) SDS prior to electrophoresis. There- VOL. 176, 1994 HEME A BIOSYNTHESIS IN BACILLUS SUBTILIS 6667

DEAE-Sephacel Sephacryl S-300 Hydroxylapatite

3- 400 2 z -300I i 2 o .2001>

.1

.100 /" I 10 20 30 40 50 10 20 30 4 snn*uu 0.6 00 * b c 14 bJ31 I . 400 C2 0.4- -300 c) ,0 Mto_ -2001 0 0.2- <~~~~~~~~~~~~~~I" k~oo

1.0 210 3.0 5'0 10 20 30 40 10 20 30 40 50 60 Fraction number FIG. 3. Chromatography profiles of the purification of B. subtilis cytochrome b-CTA from isolated membranes of B. subtilis 3G18sdhAl12/ pCTA1303 (top two panels) and of E. coli JM109/pCIA1900 (bottom three panels). Cytochrome in different fractions was analyzed by low-temperature light absorption spectroscopy and by analytical HPLC. The bar in each panel marks the fractions with cytochrome b-CTA that were pooled and used for further purification or analysis. Proteins from E. coli JM109/pCTA1900 bound to the hydroxylapatite were eluted using first a two-step gradient of 200 mM sodium phosphate (a) and 300 mM sodium phosphate (b) and then a linear gradient of 300 to 500 mM sodium phosphate (c). The phosphate buffers were in 20 mM MOPS/HCl buffer (pH 7.4) with 0.1% Thesit. fore, samples were treated with SDS at 40°C for 30 min before membranes (20). Partial digestion of the cytochrome (in SDS-PAGE. Thesit) with trypsin resulted in two larger polypeptide frag- Cytochrome b-CTA in Thesit was bound to detergent mi- ments, both of which had molecular masses of about 15 kDa, celles and eluted in a monodisperse state from gel filtration and smaller polypeptide fragments as determined by SDS- columns (a Pharmacia Sephacryl S-300 column and a Beckman PAGE (gel not shown). N-terminal sequence analyses of the SEC 4000 HPLC column with 0.1% [wt/vol] Thesit in the two 15-kDa tryptic fragments blotted to a PVDF membrane elution buffer) at an apparent size between that of the B. gave only one sequence, and it was unambiguous: FFPELNPA. subtilis cytochrome b-558 subunit (23 kDa) and that of the This sequence corresponds to residues 46 to 53 in the deduced intact B. subtilis succinate-menaquinone reductase (120 kDa) amino acid sequence of B. subtilis CtaA (Fig. 5). The sequence (data not shown). These results are in agreement with a mass is located in a hydrophilic segment and is preceded by an of about 34 kDa for cytochrome b-CTA; however, it cannot be arginine residue in the intact CtaA protein, consistent with a concluded whether the protein is monomeric or oligomeric in trypsin cleavage site. detergent. Chemical composition. The amino acid composition of The structural gene for cytochrome b-CTA is ctaA. Attempts cytochrome b-CTA purified from B. subtilis 3G18sdhAl12/ to determine the N-terminal sequence of purified cytochrome pCTA1303 and E. coli JM109/pCTA1900 was found to be b-CTA by Edman degradation were unsuccessful. The analyses similar and in reasonable agreement with that expected for the were tried both after applying the purified protein in solution CtaA protein (Table 4). Quantitative amino acid analyses directly to the sequencer and after electroblotting to PVDF indicated that protein determinations by the procedure de-

TABLE 3. Purification of B. subtilis cytochrome b-CTA from B. subtilis 3G18A12/pCTA1303 and E. coli JM109/pCTA1900 membranes B. subtilis E. coli

Purification step Protein Protoheme IX Heme A Protoheme IX/ Protein Protoheme IX Protoheme IX/ (mg)a (nmol) (nmol) protein (nmol/mg) (mg)a (nmol) protein (nmol/mg) Membranes 572 PqDb ND 760 ND Solubilization 473 426 301 0.9 435 702 1.6 DEAE-Sephacelc 122 100 87 0.8 110 246 2.2 Sephacryl S-300c 14 31 3.4 2.2 24 87 3.6 Hydroxylapatitec" 2.4 15 1.2 6.2 5.1 38 7.5

a Determined by the Lowry procedure with bovine serum albumin as the standard. b ND, not determined. c Fractions pooled as shown in Fig. 3. d Calculated values, since only part of the Sephacryl S-300 pool was loaded on the hydroxylapatite column. 6668 SVENSSON AND HEDERSTEDT J. BAcrERIOL.

B. subtilis E. coli sdhAl2/pCTA1 303 JM109/pCTA1900

1 2 3 4 5 1 2 3 4 5 Out A-| A-

B- B- C- C- D-

FIG. 5. Model of the B. subtilis CtaA polypeptide in the cytoplas- I mic membrane. Putative transmembrane a-helical segments are la- beled 1 to 8. In, the cytoplasmic (negative) side of the membrane. residues (H) that are conserved in CtaA of B. subtilis and B. 4 firnus OF4 are marked. The arrow shows a trypsin cleavage site 1 identified in this work.

F- iron center in the polypeptide (Table 5). Only trace amounts of copper were found. Spectral properties of cytochrome b-CTA. Light absorption spectra of oxidized and reduced cytochrome b-CTA isolated from B. subtilis 3G18sdhA12/pCTA1303 and E. coli JM109/ pCTA1900 were similar. At room temperature and in the FIG. 4. Purification of cytochrome b-CTA from B. subtilis oxidized state, the cytochrome showed absorption maxima at 3G18sdhAl12/pCTA1303 and E. coli JM109/pCTA1900 as analyzed by 280 nm from protein and at 414 nm from heme. The absorp- SDS-PAGE. The arrows indicate the 23,000-Mr cytochrome polypep- tion ratio, 280 to 414 nm, was about 1 in the purest prepara- tide. Lanes: 1, size markers; 2, Thesit-solubilized membrane proteins; tions. Dithionite-reduced cytochrome at room temperature 3, DEAE-Sephacel pool; 4, Sephacryl S-300 pool; 5, hydroxylapatite showed absorption maxima at 428, 528, and 558 nm (Fig. 6). pool, i.e., purified cytochrome b-CTA. In lane 5 of the left panel, 6 ,ug of protein and 37 pmol of protoheme IX were loaded, whereas in the These peaks are typical for a low-spin b-type cytochrome. An corresponding lane of the right panel, 5.5 ,ug of protein and 41 pmol of absorption peak with a maximum at about 590 nm, seen in the protoheme IX were loaded. The size markers were as follows: A, P-galactosidase (130,000); B, bovine serum albumin (68,000); C, catalase (57,500); D, fumarase (48,500); E, carbanhydrase (29,000); F, TABLE 4. Amino acid composition of isolated cytochrome b-CTAa (17,000); G, lysozyme (14,300). The samples correspond to those in Table 3. Composition (mol%) Amino acidb From B. subtilisl From E. colil CtaA pCTA1303 pCrA1900 Asx 6.2 6.6 2.7 scribed by Lowry et al., with bovine serum albumin as the Thr 5.4 5.2 4.7 standard, somewhat underestimated the CtaA protein concen- Ser 7.4 7.5 8.0 tration. Glx 6.5 8.0 6.4 Isolated cytochrome b-CTA was analyzed for heme by the Pro 3.4 4.3 3.0 pyridine hemochromogen spectrum and by reversed-phase Gly 8.2 9.2 7.0 HPLC of extracted heme. About 7 nmol of protoheme IX per Ala (18.2) 10.0 9.4 mg of protein was present in pure preparations (Table 3). This Cys 0.8 1.3 2.3 corresponds to a stoichiometry of about 0.2 mol of protoheme Val 6.7 6.1 6.7 2.2 2.4 2.7 per IX was the Met IX mol of 34-kDa polypeptide. Protoheme Ile 7.8 6.6 11.1 dominant type of heme in pure cytochrome preparations from Leu 10.4 11.4 15.1 both B. subtilis and E. coli. Some heme A (5 to 10 mol% Tyr 2.7 3.0 2.7 compared with protoheme IX) was present in all preparations. Phe 5.5 5.7 8.1 A trace amount of heme 0 was also detected in some His 1.7 2.5 3.0 preparations from E. coli; however, it constituted less than 5 Lys 5.4 5.0 3.4 mol% of the total heme content. Arg 2.8 5.0 3.1 One preparation of cytochrome b-CTA was also analyzed a The amino acid composition of CtaA is derived from the B. subtilis ctaA for iron and copper. All of the iron in the sample was DNA sequence (21). accounted for by heme, indicating that there is no nonheme b Trp not included. VOL. 176, 1994 HEME A BIOSYNTHESIS IN BACILLUS SUBTILIS 6669

TABLE 5. Chemical composition of CtaA-cytochrome b-CTA bound enzyme that reduces menaquinone (13, 15). Succinate isolated from B. subtilis 3G18sdhA12/pCTA1303a (succinate-fumarate couple [Em,7 = +30 mV]) partially re- Stoichiometry duced the cytochrome in 3G18/pCTA1303 membranes. These Component" Content (jxM) (mol/mol of results demonstrate that cytochrome b-CTA in the B. subtilis CtaA) membrane can communicate with the respiratory chain (mena- quinone-menaquinol pool). CtaA polypeptide 33c 1 B. subtilis cytochrome b-CTA in membranes Protoheme IX 5.0 0.15 In contrast, Heme A 0.4 0.01 isolated from E. coli JM109/pCTA1900 grown in LB medium Iron 5.6 0.17 was not reduced by 20 mM succinate, however, 10 mM Copper 1.4 0.04 ascorbate plus 1 mM TMPD reduced it to about 50% (spectra not shown). Taken together, the results with glycerol-3-phos- a The preparation analyzed was that used for lane 5 in Fig. 4. phate, succinate, and ascorbate-TMPD as reductants indicate b Amounts of polypeptide, heme, and metals were determined by quantitative amino acid analysis (Table 4), by the pyridine hemochromogen, and by atomic that the midpoint reduction potential of cytochrome b-CTA is absorption spectroscopy, respectively. greater than 0 mV. I This protein content was as determined by amino acid analysis assuming that the sample was pure CtaA. The protein content as determined by the procedure described by Lowry et al. was 23.8 ,uM. DISCUSSION Heme A is uniquely found in terminal oxidases of the respiratory chain in many aerobic organisms. It is synthesized difference (reduced minus oxidized) spectrum (Fig. 6, inset), from protoheme IX in a still tentative pathway in which heme results from heme A in the preparation. Neither oxidized nor O seemingly is a stable intermediate (37). The biosynthetic reduced cytochrome showed detectable absorption in the 650- step from heme 0 to heme A is a monooxygenation and an to 800-nm region (spectra not shown). The difference (reduced oxidation of the methyl group on porphyrin ring D (on carbon minus oxidized) extinction coefficient was determined to be 18), resulting in a formyl group. This reaction is analogous to 21.2 mM-1 cm-1 using the wavelength pair 558 nm minus 570 that in which chlorophyll a is converted to chlorophyll b; nm (isobestic point). however, in this latter case the modification of a methyl to a The low-temperature light absorption spectrum of dithio- formyl group occurs on ring B. For chlorophyll b, it is known nite-reduced cytochrome b-CTA isolated from B. subtilis from 1 02-labeling experiments in plants that the formyl 3G18sdhA12/pCTA1303 and E. coli JM109/pCTA1900 showed atom is derived from dioxygen (26, 36). The enzyme the split a-peak with maxima at 553 and 559 nm that was also responsible for the modification is probably a monooxygenase, seen with the membrane-bound cytochrome (Fig. 1 and 2). and a hydroxy intermediate has been postulated in the pathway Cytochrome b-CTA in membranes from B. subtilis 3G18sdh from chlorophyll a to b (2). A similar monooxygenase is A12/pCTA1303 grown in NSMP supplemented with 0.5% expected in heme A biosynthesis. (wtlvol) glycerol was completely reduced at pH 7.4 by 20 mM Genetic evidence and in vivo experimental data with E. coli glycerol-3-phosphate (glycerol-3-phosphate-dihydroxyacetone strongly suggest that the B. subtilis ctaA gene product is an phosphate redox couple [Em7 = -190 mV]). Glycerol was enzyme with a specific function in heme A synthesis from heme added to the growth medium to induce synthesis of glycerol- 0. Mutants defective in ctaA have a pleiotropic phenotype in 3-phosphate dehydrogenase, which is a peripheral membrane- that they lack two genetically and structurally distinct heme A-containing oxidases, namely, cytochrome aa3 and cyto- chrome caa3 (21, 22, 37, 39). ctaA-defective mutants can synthesize heme 0 (37). Furthermore, the B. subtilis ctaA gene on plasmids in E. coli results in accumulation of heme A in the membrane fraction (37); E. coli can synthesize heme 0, but 5060015 normally not heme A. 400 In this paper, we describe the purification of the B. subtilis CtaA protein after overexpression of the ctaA and ctaB genes g ( in both B. subtilis and E. coli. CtaA was found to be a novel FG 6 membrane-bound heme-containing protein, which we call cy- tochrome b-CTA. Tentatively, we identify it as a cytochrome b, .0 since it contains protoheme IX and shows light absorption < spectral properties typical for a low-spin cytochrome b. Cyto- chrome b-CTA in the B. subtilis membrane was found to be reduced via the respiratory chain (menaquinol pool), indirectly showing that electron transfer can occur between respiratory components and the cytochrome. A bona fide classification of CtaA as a cytochrome b requires knowledge of its specific 400 500 600 function and perhaps also its mechanism of action. Cyto- Wavelength (nm) chrome b-CTA can be only a minor cytochrome in wild-type cells but is the dominating cytochrome b in light absorption FIG. 6. Light absorption spectra at room temperature of isolated spectra of B. subtilis 3G18sdhA12/pCTA1303 membranes (Fig. B. subtilis cytochrome b-CTA. Shown are the spectra of oxidized (as b-CTA contained about 0.2 molecule isolated) (dashed line) and dithionite-reduced (solid line) cytochrome 1). Isolated cytochrome purified from membranes of B. subtilis 3G18sdhAl12/pCTA1303. The of protoheme IX per CtaA polypeptide. The cytochrome inset shows the difference (dithionite reduced minus oxidized) spec- b-CTA content in membranes of B. subtilis 3G18sdhA12/ trum. The vertical bars indicate the absorbance scales. The cuvette pCTA1303 was about 0.9 nmol per mg of membrane protein, contained 2.5 ,uM protoheme IX and was from the same preparation as calculated from the room temperature difference (reduced as that analyzed in Fig. 4 (lane 5 of the left panel). minus oxidized) absorption spectrum using the coefficient 21.1 6670 SVENSSON AND HEDERSTEDT J. BACTERIOL.

mM-1 (558 minus 570 nm) determined from the isolated 6, and 8, respectively. One or several of these His residues cytochrome. The spectrum was subtracted by that of 3G18sdh probably serve as axial heme iron ligands. A12 before the calculation in order to remove the background One urgent question is to establish the exact function of cytochrome absorption. Assuming one protoheme IX molecule CtaA in heme A synthesis. An attractive hypothesis based on per membrane-bound 34-kDa CtaA polypeptide, cytochrome the available data is that CtaA is both the monooxygenase and b-CTA would constitute about 3% of the total membrane the dehydrogenase postulated in heme A biosynthesis from protein. This amount of CtaA polypeptide seems reasonable, heme 0. Monooxygenases and dehydrogenases usually contain as judged from the purification data shown in Table 3 and the one or more prosthetic groups: flavins or metal centers (cf. SDS-PAGE pattern shown in Fig. 4. These data suggest that references 11 and 43). The isolated B. subtilis CtaA protein membrane-bound cytochrome b-CTA contains at least one does not contain flavin or nonheme iron. If CtaA is a mono- protoheme IX per CtaA polypeptide. During purification of oxygenase and provided that nonheme cofactors have not been integral membrane-bound b-type cytochromes, heme is often completely lost during purification, it seems that protoheme IX lost to some extent (cf. reference 31). This may explain the in cytochrome b-CTA plays the role of as in the P450 substoichiometric amounts of heme in the cytochrome b-CTA family of enzymes and that menaquinol functions as an elec- preparations. However, incubation of purified cytochrome tron donor to cytochrome b-CTA in the membrane. However, b-CTA with a 10-fold excess of hemin did not increase the CtaA would not be a typical P450 enzyme, since it lacks the amount of tightly bound heme (data not shown). An alterna- signature sequence FXXGXXXCXG (23) and its carbon mon- tive explanation for the low heme content is that CtaA is not a oxide difference spectrum does not show a peak in the 450-nm typical cytochrome and that the protoheme IX present in the region. purified protein is in fact substrate, rather than a prosthetic group, or is both substrate and prosthetic group, as is the case ACKNOWLEDGMENTS for the heme-degrading enzyme heme oxygenase (38). We are grateful to Karin Tsiobanelis for expert technical assistance, In B. subtilis, we have been able to detect cytochrome b-CTA to Matti Saraste for stimulating discussions on the CtaA sequence, and only in membranes from cells containing ctaA and ctaB on a to Claes von Wachenfeldt for making Fig. 5. plasmid; the plasmid used in the present work has a copy This work was supported by grants from the Swedish Natural number of about 5. Despite the presence of ctaA and ctaB in Science Research Council, the Swedish Medical Research Council, Emil och Wera Cornells Stiftelse, and Per-Eric och Ulla Schybergs the chromosome, neither gene alone on the plasmid resulted in Stiftelse. spectroscopically detectable amounts of the cytochrome in the membrane. Experiments in E. coli confirmed that both genes, REFERENCES in cis or in trans, are required for the production of B. subtilis 1. Barett, J. 1956. The prosthetic group of cytochrome a2. Biochem. cytochrome b-CTA (Table 2). Thus, both genes apparently J. 64:626-639. have to be amplified in the cell in order to obtain (over)pro- 2. Beale, S. I., and J. D. Weinstein. 1990. metabolism in duction of cytochrome b-CTA. At present, we have no expla- photosynthetic organisms, p. 287-391. In H. A. Dailey (ed.), nation for why ctaB is required for production of cytochrome Biosynthesis of heme and . McGraw-Hill Publishing b-CTA; however, one possibility is that the cytochrome is an Co., New York. experimental 3. Chepuri, V., and R. B. Gennis. 1990. The use of gene fusion to artifact in the sense that it may be formed, or determine the topology of all subunits of the cytochrome o stabilized, only under special conditions, such as when both terminal oxidase complex of Escherichia coli. J. Biol. Chem. 265: genes are amplified. 12978-12986. From its DNA sequence, B. subtilis CtaA is predicted to 4. Chepuri, V., L. Lemieux, D. C.-T. Au, and R. B. Gennis. 1990. The consist of 306 amino acid residues; the calculated mass includ- sequence of the cyo operon indicates substantial structural simi- ing the N-terminal Met residue is 34,084 Da (21). Cytochrome larities between the cytochrome o ubiquinol oxidase of Escherichia b-CTA behaves as an integral membrane protein, i.e., it is coli and the aa3-type family of cytochrome oxidases. J. Biol. Chem. tightly membrane bound, and when isolated, it requires deter- 265:11185-11192. 5. Falk, J. E. 1964. Biochemica et Biophysica Acta Library, vol. 2, p. gent to remain soluble in aqueous solutions. On the basis of 181-182. Elsevier Publishing B. V., Amsterdam. hydropathy profiles and the distribution of charged residues 6. Friden, H., M. R. Cheesman, L. Hederstedt, K. K. Andersson, and (40), the CtaA polypeptide is a multitopic membrane protein A. J. Thomson. 1990. Low temperature EPR and MCD studies on with eight transmembrane at-helical segments (Fig. 5). The cytochrome b-558 of the Bacillus subtilis succinate:quinone oxi- discrepancy between the mass of isolated CtaA estimated by doreductase indicate bis-histidine coordination of the heme iron. SDS-PAGE (23 kDa) and that predicted from the sequence Biochim. Biophys. Acta 1041:207-215. (34 kDa) can be explained by the hydrophobic character of the 7. Friden, H., L. Hederstedt, and L. Rutberg. 1987. Deletion of the protein or by posttranslational processing. Bacillus subtilis sdh operon. FEMS Microbiol. Lett. 41:203-206. The N- and C-terminal halves of the CtaA polypeptide, each 8. Friden, H., L. Rutberg, K. Magnusson, and L. Hederstedt. 1987. Genetic and biochemical characterization of Bacillus subtilis mu- of which has four predicted transmembrane segments, show tants defective in expression and function of cytochrome b-558. 28% overall sequence identity (residues 1 to 153 aligned to Eur. J. Biochem. 168:695-701. residues 154 to 306 using the GAP program). This internal 9. Hagerhall, C., R Aasa, C. von Wachenfeldt, and L. Hederstedt. sequence similarity suggests that the polypeptide has origi- 1992. Two in Bacillus subtilis succinate:menaquinone oxi- nated as the result of a tandem gene duplication. Cys and His doreductase (complex II). Biochemistry 31:7411-7421. residues often function as ligands to metal centers in proteins. 10. Haima, P., S. Bron, and G. Venema. 1987. The effect of restriction One Cys residue in each of the two predicted large externally on shotgun cloning in Bacillus subtilis Marburg. Mol. Gen. Genet. oriented extramembrane domains is also conserved (Fig. 5). 209:.335-342. These two Cys residues seem functionally important 11. Harayama, S., M. Kok, and E. L. Neidle. 1992. Functional and since they evolutionary relationship among diverse oxygenases. Annu. Rev. are also found in CtaA from B. firnus OF4 (27), which shows Microbiol. 46:565-601. 38% sequence identity to B. subtilis CtaA. Among the seem- 12. Hederstedt, L. 1986. Molecular properties, genetics and biosyn- ingly conserved residues in B. subtilis CtaA are His-60, His-123, thesis of Bacillus subtilis succinate dehydrogenase complex. Meth- His-216, and His-278 in predicted transmembrane helices 2, 4, ods Enzymol. 126:399-414. VOL. 176, 1994 HEME A BIOSYNTHESIS IN BACILLUS SUBTILIS 6671

13. Hederstedt, L. Unpublished data. Biol. Chem. 268:26927-26934. 14. Hoch, J. A. 1991. Genetic analysis in Bacillus subtilis. Methods 30. Saiki, K., T. Mogi, K. Ogura, and Y. Anraku. 1993. In vitro heme Enzymol. 204:305-320. O synthesis by the cyoE gene product from Escherichia coli. J. Biol. 15. Holmberg, C., and B. Rutberg. 1991. Expression of the gene Chem. 268:26041-26044. encoding glycerol-3-phosphate dehydrogenase (glpD) in Bacillus 31. Salerno, J. C., J. P. McCurley, J. H. Dong, M. R. Doyle, and C. A. subtilis is controlled by antitermination. Mol. Microbiol. 5:2891- Yu. 1986. The EPR spectra of the cytochrome b-cl complex of 2900. Rhodopseudomonas sphaeroides. Biochem. Biophys. Res. Com- 16. James, W. S., F. Gibson, P. Taroni, and R K. Poole. 1989. The mun. 136:616-621. cytochrome oxidases of Bacillus subtilis: mapping of the gene 32. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular affecting cytochrome aa3 and its replacement by cytochrome o in a cloning: a laboratory manual. Cold Spring Harbor Laboratory, mutant strain. FEMS Microbiol. Lett. 58:277-282. Cold Spring Harbor, N.Y. 17. Laureus, M., T. Haltia, M. Saraste, and M. Wikstrom. 1991. 33. Santana, M., F. Kunst, M. F. Hullo, G. Rapaport, A. Danchin, and Bacillus subtilis expresses two kinds of haem-A-containing termi- P. Glaser. 1992. Molecular cloning, sequencing and physiological nal oxidases. Eur. J. Biochem. 197:699-705. characterization of the qox operon from Bacillus subtilis encoding 18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. the aa3-600 quinol oxidase. J. Biol. Chem. 267:10225-10231. 1951. Protein measurement with the Folin phenol reagent. J. Biol. 34. Saraste, M., T. Metso, T. Nakari, T. Jalli, M. Laureus, and J. van Chem. 193:265-275. der Oost. 1991. The Bacillus subtilis cytochrome c oxidase: varia- 19. Mandel, M., and A. Higa. 1970. Calcium-dependent bacteriophage tion on a conserved protein theme. Eur. J. Biochem. 195:517- DNA infection. J. Mol. Biol. 53:159-162. 525. 20. Matsudaira, P. 1990. Limited N-terminal sequence analysis. Meth- 35. Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl ods Enzymol. 182:602-613. sulphate-polyacrylamide gel electrophoresis for separation of pro- 21. Mueller, J. P., and H. W. Taber. 1989. Isolation and sequence of 1 to 100 ctaA, a gene required for cytochrome aa3 biosynthesis and sporu- teins in the range from kDa. Anal. Biochem. 166:368- lation in Bacillus sublilis. J. Bacteriol. 171:4967-4978. 379. 22. Mueller, J. P., and H. W. Taber. 1989. Structure and expression of 36. Schneegurt, M. A., and S. I. Beale. 1992. Origin of the chlorophyll the cytochrome aa3 regulatory gene ctaA of Bacillus subtilis. J. b formyl oxygen in Chlorella vulgaris. Biochemistry 31:11677- Bacteriol. 171:4979-4986. 11683. 23. Nelson, D. R., T. Kamataki, D. J. Waxman, F. P. Guengerich, 37. Svensson, B., M. Lubben, and L. Hederstedt. 1993. Bacillus subtilis R. W. Estabrook, R. Feyereisen, F. J. Gonzalez, M. J. Coon, I. C. CtaA and CtaB function in haemA biosynthesis. Mol. Microbiol. Gunsalus, 0. Gotoh, K. Okuda, and D. W. Nebert. 1993. The P450 10:193-201. superfamily: update on new sequences, gene mapping, accession 38. Takahashi, S., J. Wang, D. L. Rousseau, K. Ishikawa, T. Yoshida, numbers, early trivial names of enzymes and nomenclature. DNA J. R Host, and M. Ikeda-Saito. 1994. Heme-heme oxygenase Cell Biol. 12:1-51. complex. J. Biol. Chem. 269:1010-1014. 24. Nobrega, M. P., F. G. Nobrega, and A. Tzagalof. 1990. COX1O 39. van der Oost, J., C. von Wachenfeldt, L. Hederstedt, and M. codes for a protein homologous to the ORF1 product of Paracoc- Saraste. 1991. Bacillus subtilis cytochrome oxidase mutants: bio- cus denitrificans and is required for the synthesis of yeast cyto- chemical analysis and genetical evidence for two aa3-type oxidases. chrome oxidase. 1. Biol. Chem. 265:14220-14226. Mol. Microbiol. 8:2063-2072. 25. Petricek, M., L. Rutberg, and L. Hederstedt. 1989. The structural 40. von Heijne, G. 1992. Membrane protein structure prediction. gene for aspartokinase II in Bacillus subtilis is closely linked to the Hydrophobicity analysis and the positive-inside rule. J. Mol. Biol. sdh operon. FEMS Microbiol. Lett. 61:85-88. 225:487-494. 26. Porra, R. J., W. Schafer, E. Cmiel, I. Katheder, and H. Scheer. 41. von Wachenfeldt, C., and L. Hederstedt. 1992. Molecular biology 1993. Derivation of the formyl-group oxygen of chlorophyll b from of Bacillus subtilis cytochromes. FEMS Microbiol. Lett. 100:91- molecular oxygen in green leaves of a higher plant (Zea mays). 100. FEBS Lett. 323:31-34. 42. Wu, W., C. K. Chang, C. Varotsis, G. T. Babcock, A. Puustinen, 27. Quirk, P. G., D. B. Hicks, and T. A. Krulwich. 1993. Cloning of the and M. Wikstrom. 1992. Structure of the heme 0 prosthetic group cta operon from alkaliphilic Bacillus firmus OF4 and characteriza- from the terminal quinol oxidase ofEschenichia coli. J. Am. Chem. tion of the pH-regulated cytochrome aa3 oxidase it encodes. J. Soc. 114:1182-1187. Biol. Chem. 268:678-685. 43. Yamamoto, S., and Y. Ishimura. 1991. Dioxygenases and mo- 28. Ratio, M., T. Jalli, and M. Saraste. 1987. Isolation and analysis of nooxygenases, p. 315-344. In J. Kuby (ed.), A study of enzymes. the genes for cytochrome oxidase in Paracoccus denitrificans. CRC Press, Boca Raton, Fla. EMBO J. 6:2825-2833. 44. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved 29. Saiki, K., T. Mogi, H. Hori, M. Tsubaki, and Y. Anraku. 1993. M13 phage cloning vectors and host strains: nucleotide sequence Identification of the functional domain in heme 0 synthase. J. of the M13mpl8 and pUC19 vectors. Gene 33:103-119.