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Journal of Bacteriology, December 2005, p. 8361-8369, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8361-8369.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Heme A Synthase Enzyme Functions Dissected by Mutagenesis of Bacillus subtilis CtaA

Lars Hederstedt,* Anna Lewin, and Mimmi Throne-Holst{dagger}

Department of Cell & Organism Biology, Lund University, Sölvegatan 35, SE-223 62 Lund, Sweden

Received 15 August 2005/ Accepted 13 September 2005


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ABSTRACT
 
Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.


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INTRODUCTION
 
Heme A is a highly specialized variant of heme that is important for cellular aerobic respiration and energy conversion in animals, plants, and microorganisms. It is a prosthetic group in the mitochondrial cytochrome c oxidase and in similar oxidases of bacterial aerobic respiratory chains. Heme A is synthesized from heme B (protoheme IX) with heme O as a stable intermediate (15). Heme O is derived from heme B by substitution of the vinyl group on carbon 2 of the porphyrin ring (Fisher nomenclature) with a hydroxyethyl farnesyl side group. Heme A differs from heme O by containing a formyl group instead of a methyl group on carbon 8 of the porphyrin ring.

All known heme A-containing oxidases belong to the heme-copper superfamily of terminal oxidases (6). The gram-positive bacterium Bacillus subtilis has two heme A-containing terminal oxidases, cytochrome aa3 and cytochrome caa3. The polypeptide constituents of these two oxidases are encoded by the qoxABCD operon and the ctaCDEF gene cluster, respectively (30). Cytochrome aa3 is the major type a cytochrome under most growth conditions (32). Expression of cytochrome caa3 is catabolite repressed; cells grown in nutrient sporulation medium supplemented with phosphate (NSMP) contain 10-fold more of this oxidase compared to cells grown in NSMP supplemented with 0.5% glucose (1). Heme O and heme A synthase are encoded in B. subtilis by the ctaB and ctaA genes, respectively (16, 24). These genes are located immediately upstream of the ctaCDEFG gene cluster at 133o on the circular chromosomal map. There is also a second heme O synthase gene, ctaO (yjdK), located at 109o on the chromosome, but this gene is not essential for heme O synthesis (28). The ctaB gene is cotranscribed with ctaCDEF (13), and ctaA is transcribed in the opposite direction (Fig. 1A). The promoter regions for ctaA and ctaB overlap (34). CtaA and CtaB are integral polytopic membrane proteins (15, 25). B. subtilis cells can grow aerobically in the absence of heme O and heme A. CtaA- and CtaB-deficient mutants are viable because of the presence of a cytochrome bd terminal respiratory oxidase (32). This property makes B. subtilis ideal for experimental studies of heme A synthesis and cytochrome a biogenesis.



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  		FIG. 1. Maps of the genes and DNA constructs used in this work. (A) The cta region in the B. subtilis chromosome. Two promoters present between ctaA and ctaB are indicated by bent arrows. (B) The DNA fragment used to delete the chromosomal ctaA gene by homologous recombination, resulting in strain LMT20R. EcoRI and BamHI restriction sites added via PCR are indicated. (C) The ctaA gene in pCTHI10. The position of an introduced HindIII restriction site and a sequence (seq.) resulting in a His6 tag at the C terminus of the CtaA polypeptide are indicated.

From studies of Escherichia coli cells expressing B. subtilis ctaA or Bacillus stearothermophilus ctaA, it has been demonstrated that the CtaA protein catalyzes heme A synthesis from heme O (3, 20, 26). Recently, Hegg and collaborators, using the B. subtilis ctaA gene expressed in E. coli, showed that heme A synthase activity is dependent on the presence of molecular oxygen and presented evidence that hydroxymethyl heme O (heme I) is a reaction intermediate (3). It is thought that the methyl side chain on carbon 8 of the porphyrin ring of heme O is subjected to two consecutive hydroxylations catalyzed by the heme A synthase. The second hydroxylation would result in an unstable dihydroxymethyl group, which spontaneously decomposes into a formyl group and a water molecule. Isotope labeling experiments suggest that the oxygen atom of the formyl group is not directly derived from molecular oxygen (4). This is unexpected, and notably, B. subtilis heme A synthase does not have the biochemical or amino acid sequence characteristics of a P450 type of enzyme, which contains heme as a prosthetic group and activates molecular oxygen (25). The molecular mechanism of catalysis by heme A synthase remains to be elucidated and might include unprecedented features. Also, little is known about the chemical composition and structure of heme A synthase. Furthermore, the CtaA protein might play a direct role in the assembly of cytochrome a by mediating heme A insertion into the QoxA and CtaD proteins in the B. subtilis membrane.

By using recombinant B. subtilis strains that overproduce CtaA, two types of preparations which differ in heme composition have been isolated (24), i.e., cyt b-CTA containing heme B and small amounts of heme A and cyt ba-CTA containing about equal amounts of heme B and heme A. The iron atoms of heme B and heme A in isolated oxidized CtaA are low spin (electron paramagnetic resonance gmax signals at 3.7 and 3.5, respectively) (24). Heme A (Em,7 = +242 mV) bound to isolated CtaA is thought to be the enzyme product waiting to be used in the synthesis of heme A-containing terminal oxidases. It has been suggested that bound heme B (Em,7 = +85 mV) is a prosthetic group of CtaA with an electron transfer function within the enzyme. However, only substoichiometric amounts of heme B (≤0.4 mol of heme per mol of CtaA polypeptide) have been found in isolated CtaA (25). The possibility cannot be ruled out that the presence of heme B, or the substoichiometric amount of heme, is an experimental artifact resulting from overproduction of CtaA polypeptide. Less than 0.02 mol of copper and non-heme iron atoms per mol of polypeptide has been found in isolated B. subtilis CtaA (25). In the absence of cofactors other than heme, it is reasonable to assume that heme iron is part of the catalytic center of heme A synthase. Heme O can thus be both a substrate and a cofactor for the enzyme, similar to the situation with heme oxygenase, where heme B plays these two roles (19).

Based on the hydropathy profile (11), the positive-inside rule (29), and topology analysis by fusions to alkaline phosphatase (our own unpublished data), the CtaA polypeptide is predicted to have eight membrane-spanning {alpha}-helical segments and the N terminus exposed on the negative side of the membrane (Fig. 2). Genes encoding proteins similar to CtaA are present in various organisms, such as eubacteria, archaea, yeast, and humans (18, 27). The CtaA orthologues contain nine highly invariant residues. Four of these are histidine residues, His-60, His-123, His-216, and His-278, in the B. subtilis protein (Fig. 2). These residues are all predicted to be located close to the positive (extracytoplasmic) side of the membrane. Histidine residues often function as axial ligands to heme iron in proteins. To analyze the functional roles of the four invariant histidine residues in B. subtilis CtaA, we have changed each of them individually to a methionine and a leucine residue. These particular replacements were chosen because they have been successful in studies of other membrane-bound cytochromes (14). A methionine, but not a leucine, residue can substitute for a histidine residue as an axial heme ligand. We report here on the in vivo enzyme activities and heme contents of the different mutant variants of CtaA which were overproduced in B. subtilis.



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  		FIG. 2. Topology model of the CtaA protein in the cytoplasmic membrane of B. subtilis. The eight predicted membrane-spanning {alpha} helices are numbered I to VIII. The outer side (+) and cytoplasmic side (–) of the membrane and the approximate location of four histidine residues that are invariant in CtaA orthologues are indicated.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and oligonucleotides. The strains and plasmids used in this work are listed in Table 1. The oligonucleotides used for PCR, mutagenesis, and DNA sequence analysis are presented in Table 2.


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  		TABLE 1. Bacterial strains and plasmids used in this study


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  		TABLE 2. Oligonucleotides used as primers in this work

Media and general growth of bacteria. B. subtilis strains were kept on tryptose blood agar base (TBAB; Difco Co.) plates supplemented with 0.5% (wt/vol) glucose. For the preparation of membranes, B. subtilis strains were grown in NSMP, pH 7.0, containing 0.5% (wt/vol) glucose. Membranes for cytochrome c oxidase activity measurements were prepared from cells grown in NSMP without glucose. Antibiotics were used at the following concentrations when appropriate: B. subtilis, chloramphenicol at 3 to 5 mg/liter and spectinomycin at 150 mg/liter; E. coli, ampicillin at 125 mg/liter and tetracycline, kanamycin, and chloramphenicol at 12.5 mg/liter.

General methods. B. subtilis was grown to competence essentially as described by Hoch (9). N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) oxidation by colonies on TBAB and NSMP plates was assayed as described before (1, 12). Membranes were isolated from B. subtilis strains as described previously (8) and stored at –80°C. Light absorption spectroscopy (22) and cytochrome c oxidase activity measurements (5) were performed as previously described. Protein concentrations were determined by the bicinchoninic acid protein assay (Pierce Chemical Co.) with bovine serum albumin as the standard. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was done by the method of Schägger and von Jagow (21). Immunoblotting (Western blotting) was done as described before (1), with mouse anti-hexahistidyl monoclonal antibodies (Amersham Pharmacia), diluted 3,000-fold, and anti-mouse immunoglobulin G peroxidase-linked sheep antiserum (Amersham Pharmacia) diluted 1,500-fold. Heme was extracted and analyzed by reversed-phase high-performance liquid chromatography (HPLC) as described before (26), except that a Waters 996 photodiode array detector was used. PCR was carried out with the proofreading DNA polymerase kit Expand Plus (Roche Molecular Biochemicals) and a Perkin-Elmer GeneAmp PCR system 2400 reactor.

Construction of B. subtilis strain LMT20R. The ctaA gene in B. subtilis strain 1A1 was deleted by homologous double-crossover recombination with a DNA fragment spanning the ctaA chromosomal region but carrying a gene for spectinomycin resistance, spc, in place of ctaA. First, plasmid pSPC1(+/–) was constructed to obtain appropriate restriction sites on each side of the spc gene. This plasmid was obtained by moving a 1.2-kb PstI fragment from pDG1726 into pBluescript SK(–). The resulting two variant plasmids were named pSPC1(+) and pSPC(–), respectively, depending on the relative orientation of the spc gene. The orientation of the spc gene in pSPC1(+) is the opposite of that of the bla gene in pBluescript II SK(–). A 0.4-kb fragment containing the pycA-ctaA intergenic region was amplified with strain 1A1 chromosomal DNA as a template in a PCR with primers pycA1 and pycA3 (Table 2). This introduced an EcoRI and a KpnI restriction site at each end of the fragment, respectively. The amplified fragment was cut with KpnI and ligated into pBluescript II SK(–) cleaved with KpnI and HincII, resulting in plasmid pDCTA1. An EcoRI-BamHI fragment of pSPC1(–) containing the spc gene was ligated into pDCTA1, resulting in plasmid pDCTA2. A 1.5-kb fragment containing the ctaB gene was amplified by using pCTA1305 as a template in a PCR with primers ctaB6 and ctaB7 (Table 2). A BamHI restriction site was added via the ctaB7 primer. The amplified fragment was cut with BamHI and ligated to pDCTA2 cleaved with the same enzyme, resulting in a linear fragment (Fig. 1B), which was used to transform B. subtilis 1A1 to spectinomycin resistance. The resulting strain was named LMT20R, and the ctaA deletion was confirmed by PCR.

Construction of plasmids for site-directed mutagenesis and expression of His6-tagged CtaA. Transforming E. coli cells with plasmids carrying the entire B. subtilis ctaA gene, including the promoter region, resulted in accumulation ofmutations in ctaA, most likely due to a toxic effect of the CtaA protein in E.coli.Therefore, the ctaA gene was cloned as two separate fragments in pCTAAHIND1 and pCTAAHIND2. To obtain the two fragments, a HindIII site at bp 8 to 13 of the coding sequence (Fig. 1C) was introduced by changing bp 12 from an A to a T. This change in the DNA sequence does not affect the amino acid sequence of the CtaA protein but enables cloning of the major part of ctaA unexpressed in E. coli.

For the construction of pCTAAHIND1, a 0.9-kb fragment containing the ctaA gene was amplified by using pCTA1305 as the template in a PCR with primers CTAAHIA and CTAAHIND1 (Table 2), introducing an EcoRI and a HindIII restriction site at each end of the fragment. The CTAAHIA primer also introduces a sequence resulting in a His6 tag at the C terminus of CtaA. The amplified fragment was cut with EcoRI and HindIII and ligated into pHPKS, resulting in plasmid pCTAAHIND1. A 0.9-kb KpnI-BamHI fragment from pCTAAHIND1 (the KpnI site is in the vector sequence) was cloned in pALTER-1, resulting inplasmid pCTALT10, which was used as the template in site-directed mutagenesis.

For construction of pCTAAHIND2, carrying the native ctaA promoter region, a Shine-Dalgarno sequence, and the first 12 bp of the coding sequence of the ctaA gene, a 164-bp BamHI-HindIII fragment was first amplified by using pCTA1305 as a template in a PCR with primers ctaA1 and CTAAHIND2 (Table 2). The BamHI and HindIII restriction sites were added via the primers. The amplified fragment was cut with BamHI and HindIII and ligated into pHPKS, resulting in plasmid pCTAAHIND2. A 0.9-kb HindIII-SalI fragment from pCTAAHIND1 (the SalI site is in the vector sequence) was moved into pCTAAHIND2, resulting in plasmid pCTHI10.

Site-directed mutagenesis. Site-directed mutagenesis of the ctaA gene was carried out with the Altered Sites II mutagenesis kit (Promega) or by overlap extension PCR (17). In both procedures, pCTALT10 was used as a template. The different oligonucleotides used for mutagenesis are presented in Table 2. To minimize the risk of reversion, each mutation included the change of two nucleotides in the codon. Mutant ctaA DNA fragments were moved, as 0.9-kb HindIII-SalI fragments, from pCTALT10 derivatives into pCTAAHIND2, resulting in the pHxxxL and pHxxxM series of plasmids (xxx indicates the corresponding CtaA amino acid residue number; Fig. 2), which were used to transform B. subtilis LMT20R. DNA sequence analysis of the entire ctaA fragment in the pCTALT10 derivatives confirmed the desired mutations and excluded the presence of unwanted mutations.

Purification of His6-tagged CtaA. Isolated B. subtilis membranes (100 to 150 mg of protein) were solubilized at 10 to 20 mg of protein per ml in 4% (wt/vol) Thesit (Fluka GmbH) in 20 mM sodium-3-(N-morpholino)propanesulfonic acid buffer (Na-MOPS/HCl), pH 7.4, containing Complete protease inhibitor cocktail (Roche) and 0.15 M NaCl. The mixture was incubated at room temperature for 15 min and then centrifuged at 20,000 rpm (Beckman JA20 rotor) for 45 min at 4°C. The resulting supernatant was centrifuged at 59,000 rpm (Sorvall RP80-AT rotor) for 60 min at 4°C.

For affinity chromatography, the supernatant from the final centrifugation step was mixed with 2 ml of Ni-nitrilotriacetic acid matrix (QIAGEN) for 60 min at 4°C by slow mixing. The matrix mixture was then packed into a column and washed with 6 ml of 20 mM Na-MOPS/HCl buffer (pH 7.4) containing 0.15 M NaCl and 0.25% Thesit (elution buffer). Protein was eluted with 2-ml portions of elution buffer containing 5 mM, 10 mM, 50 mM, and 100 mM histidine, respectively. Fractions of 2 ml were collected and kept chilled on ice until used. His6-tagged CtaA eluted at 50 mM histidine. CtaA peak fractions were pooled and dialyzed at +4°C against 20 mM Na-MOPS/NaCl buffer, pH 7.4, with 6,000-Da cutoff dialysis membranes. Finally, the samples were concentrated with Centricon 30 devices (Millipore). The final samples were kept on an ice bath in the dark until analyzed.


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RESULTS
 
Construction and properties of a B. subtilis ctaA deletion mutant, strain LMT20R. CtaA not only catalyzes the conversion of heme O into heme A in B. subtilis, it also physically interacts with the CtaB protein (2) and possibly functions in the assembly of cytochrome aa3 and cytochrome caa3. To be able to assay these functions, we decided to produce and analyze mutant CtaA variants in B. subtilis. By this approach, we also circumvent problems associated with heterologous expression of CtaA in, e.g., E. coli such as toxicity of the protein and possible assembly defects. To have a robust experimental system, excluding problems with genetic recombination between chromosomal and plasmid-borne ctaA sequence variants, a B. subtilis strain, LMT20R, with the entire ctaA coding region deleted and replaced with a spectinomycin resistance gene (Fig. 1B) but with the rest of the cta gene cluster intact was constructed as described in Materials and Methods.

When grown under aerobic conditions, strain LMT20R was found to lack cytochromes of type a but to contain cytochrome bd, as judged by light absorption spectroscopy of isolated membranes. As expected from the absence of a functional cytochrome caa3, colonies of B. subtilis LMT20R grown on TBAB or NSMP agar plates were unable to oxidize TMPD. Membranes isolated from LMT20R grown in NSMP without glucose showed less than 0.5% cytochrome c oxidase activity compared to that of membranes from the parental B. subtilis strain 1A1 grown under the same conditions. These properties of strain LMT20R are as expected from the lack of a functional CtaA.

Complementation of LMT20R by plasmid-borne ctaA. Plasmid pCTHI10 was constructed for the production of B. subtilis CtaA polypeptide containing a hexahistidyl sequence (His6 tag) at the C-terminal end. The ctaA gene in the plasmid has its natural promoter region (Fig. 1C). Strain LMT20R containing plasmid pCTHI10 or plasmid pCTA1302, which encodes untagged CtaA, showed normal TMPD oxidation activity, cytochrome c oxidase activity, and cytochrome a content, as indicated by the absorption band at 600 nm in the difference (reduced-minus-oxidized) spectrum of membranes from cells grown in NSMP supplemented with 0.5% glucose (Fig. 3, trace A). LMT20R containing the empty plasmid vector pHPKS did not complement the cytochrome a deficiency (Fig. 3, trace J). The results confirmed that heme O synthase, CtaB, is produced in strain LMT20R and demonstrated that the His6-tagged variant of CtaA encoded by pCTHI10 is functional.



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  		FIG. 3. Difference (dithionite-reduced minus ferricyanide-oxidized) light absorption spectra of membranes from B. subtilis LMT20R carrying plasmids pCTHI10 (A), pH60L (B), pH60M (C), pH123L (D), pH123M (E), pH216L (F), pH216M (G), pH278L (H), pH278M (I), and pHPKS (J). The absorbance scale is indicated by the bar. The cuvettes contained 4 mg of membrane protein per ml, except in the case of the LMT20R/pH60L sample, which contained 2 mg of membrane protein per ml. The spectra were recorded at room temperature.

Purification and analysis of His6-tagged CtaA. By Western blot analysis with antibodies directed against the His6 tag, CtaA was detected in isolated membranes from LMT20R/pCTHI10 (Fig. 4). As expected, this antigen was not found in membranes from LMT20R/pHPKS.



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  		FIG. 4. Western blot detection of His6-tagged CtaA in membranes isolated from B. subtilis LMT20R carrying plasmids pHPKS (lane 1), pCTHI10 (lane 2), pH60L (lane 3), pH60M (lane 4), pH123L (lane 5), pH123M (lane 6), pH216L (lane 7), pH216M (lane 8), pH278L (lane 9), and pH278M (lane 10). Ten micrograms of protein was loaded into each well, and His6-tagged CtaA was detected with anti-His6 antibodies.

The His6-tagged CtaA protein was solubilized from LMT20R/pCTHI10 membranes with the nonionic detergent Thesit and purified by affinity chromatography as described in Materials and Methods. Membranes of LMT20R/pHPKS, which do not contain CtaA, and LMT20R/pCTA1302, which contain untagged CtaA, were subjected to the same purification procedure, and the samples obtained were used as controls to estimate the presence of contaminating proteins and hemes in the final preparations of purified CtaA. The His6-tagged CtaA protein purified from LMT20R/pCTHI10 is referred to in the following text as CtaA-wt, whereas the preparation from LMT20R/pHPKS is referred to as the negative control. SDS-PAGE and staining for protein showed that CtaA was the major protein in the CtaA-wt preparation (Fig. 5, lane 1). This demonstrated that His6-tagged CtaA protein can be isolated from detergent-solubilized B. subtilis membranes by the use of the affinity tag at the C terminus.



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  		FIG. 5. SDS-PAGE analysis of preparations of purified CtaA. Shown are CtaA-wt (lane 1), CtaA-H60L (lane 2), CtaA-H60M (lane 3), CtaA-H123L (lane 4), CtaA-H123M (lane 5), CtaA-H216M (lane 6), CtaA-H278L (lane 7), CtaA-H278M (lane 8), and LMT20R/pHPKS (negative control; lane 9). CtaA has a calculated mass of 34 kDa but migrates as a 23-kDa polypeptide during electrophoresis and is prone to aggregation when denatured in the presence of SDS (24, 25). The position of monomeric CtaA in the gel is indicated by an arrow, and that of aggregated CtaA is indicated by a star. About the same amount of sample, as estimated by the absorption at 280 nm, was loaded in each lane. The gel (10% acrylamide) was stained for protein with Coomassie brilliant blue.

HPLC analysis of hemes extracted from purified CtaA-wt showed heme B and a small amount of heme A (Fig. 6). The light absorption spectrum of CtaA-wt reduced with dithionite is shown in Fig. 7A. This spectrum is very similar to that of untagged CtaA (cyt b-CTA) previously purified with a combination of chromatographic methods (24), except for the weak broad absorption in the 580- to 600-nm region. Reduced heme B and heme A bound to CtaA show {alpha}-band absorption maxima at 559 nm and 585 nm, respectively (24). It has previously been shown that heme A in CtaA is fully reducible with ascorbate, whereas the heme B component is only partly reducible with ascorbate (24). CtaA-wt showed absorption maxima at 559 nm and 585 nm when reduced with 8 mM ascorbate (spectrum not shown).



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  		FIG. 6. HPLC elution profiles of heme extracted from purified CtaA protein variants CtaA-wt, CtaA-H60L, and CtaA-H216M. The positions where heme B, heme I, heme A, and heme O elute are indicated. The position of heme A is also indicated by a star in each chromatogram.



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  		FIG. 7. Light absorption spectra of purified wild-type and mutant CtaA. (A) Spectra of dithionite-reduced, purified, His6-tagged, wild-type CtaA isolated from membranes of LMT20R/pCTHI10 (wild type) and LOA10/pCTHI10 (lacks heme O). A sample obtained when a corresponding amount of membrane from LMT20R/pCTA1302 (contains CtaA without a His6 tag) was subjected to the protocol for isolation of His6-tagged CtaA served as a negative control to verify that purification of CtaA and chromophores was dependent on the tag. (B) Spectra of dithionite-reduced, purified B. subtilis wild-type CtaA (CtaA-wt; 4.5 µM heme) and mutant variants of CtaA. CtaA-H60L (6.7 µM heme), CtaA-H60M (6.2 µM heme), CtaA-H123M (4.4 µM heme), and CtaA-H216M (2.7 µM heme) are shown. The negative control (<0.5 µM heme) is a sample obtained from strain LMT20R/pHPKS, which lacks CtaA. The spectra were recorded at room temperature.

Properties of CtaA produced in a heme O-deficient strain. To determine if the weak broad absorption in the 585- to 600-nm region in spectra of dithionite-reduced samples of CtaA-wt is due to compounds derived from heme O, we analyzed the protein after production in B. subtilis strain LOA10R, which lacks the two heme O synthases CtaB and CtaO. CtaA purified from LOA10R/pCTHI10 contained only heme B, as determined by HPLC analysis (data not shown), and lacked absorption in the 585- to 600-nm spectral region (Fig. 7A). This result and the observation that samples obtained from membranes with untagged CtaA (LMT20R/pCTA1302 membranes) lacked absorption in the 500- to 650-nm region (Fig. 7A) showed that the material responsible for the absorption in the 585- to 600-nm region is bound to CtaA-wt and derived from heme O. Mutant CtaA variants lacking heme A synthase activity but containing bound heme O (see below) did not show absorption in the 585- to 600-nm region (see, e.g., CtaA-H60L in Fig. 7B). We conclude that the absorption in the 585- to 600-nm region in spectra of dithionite-reduced CtaA-wt is due to products originating from heme O and the activity of heme A synthase.

Site-directed mutagenesis of the ctaA gene and analysis of mutant CtaA. To analyze the roles of the four invariant histidine residues in B. subtilis CtaA, His-60, His-123, His-216, and His-278 (Fig. 2), we individually changed each of these residues to a methionine and a leucine. Plasmids encoding mutant CtaA were named pH60L for a mutation changing His-60 into a Leu, pH60M for a mutation changing the same His into a Met, and so on. These plasmids (Table 1) are identical to pCTHI10 (encoding CtaA-wt), except for the introduced base pair substitutions in ctaA.

B. subtilis strain LMT20R was transformed with plasmids pH60L, pH60M, pH123L, pH123M, pH216L, pH216M, pH278L, and pH278M. Membranes isolated from all of the different LMT20R transformants, except LMT20R/pH216L, contained His6-tagged CtaA (Fig. 4). Colonies of LMT20R carrying plasmid pH60M, pH278L, or pH278M were TMPD oxidation positive on NSMP plates, showing that the CtaA variants encoded by these plasmids are functional. Plasmid pH216M weakly complemented the TMPD oxidation deficiency of strain LMT20R (Table 3). The TMPD oxidation-negative phenotype of strain LMT20R was not complemented by pH60L, pH123L, or pH123M (Table 3), indicating that the CtaA variants encoded by these plasmids are not enzymatically active. As expected from the lack of CtaA protein, LMT20R/pH216L cells were TMPD oxidation negative.


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  		TABLE 3. Phenotype of B. subtilis LMT20R containing different plasmids and types of heme present in CtaA isolated from the respective strain

Membranes from LMT20R carrying pH60M, pH278L, or pH278M contained normal amounts of cytochrome a, as judged from the absorption peak at 600 nm in reduced-minus-oxidized difference spectra (Fig. 3, traces C, H, and I). In contrast, membranes from LMT20R containing pH60L, pH123L, pH123M, pH216L, or pH216M (Fig. 3, traces B, D, E, F, and G) were deficient in cytochrome a. Cytochrome bd terminal oxidase was present in membranes of all of the strains, as indicated by the absorption at about 597 nm and 627 nm in the difference spectra.

Properties of isolated mutant CtaA variants. The seven mutant variants of CtaA were purified from membranes by the same protocol as for the purification of CtaA-wt from LMT20R/pCTHI10 membranes. SDS-PAGE analysis of the preparations and staining for protein showed that they contained CtaA and little contaminating polypeptides (Fig. 5). Purified preparations of the mutant variants of CtaA are referred to in the following text as CtaA-H60L for CtaA purified from LMT20R/pH60L and so on.

Hemes were present in all samples that contained purified mutant CtaA protein. CtaA-H216M, CtaA-H278L, and CtaA-H278M contained smaller amounts of heme per CtaA protein than the other variants, as determined by the pyridine hemochromogen (data not shown). To determine which types of heme were bound to the various purified CtaA variants, heme was extracted and analyzed by HPLC (Fig. 6 and Table 3). CtaA-H60M contained heme B and heme A at levels comparable to that of CtaA-wt. Small amounts of heme B and heme A were found in CtaA-H278L and CtaA-278 M. CtaA-H216M contained heme B, heme O, and heme I (see below), as well as trace amounts of heme A. CtaA-H60L, CtaA-H123L, and CtaA-H123M contained heme B and heme O.

The light absorption spectrum of CtaA-H60M reduced with dithionite was very similar to that of CtaA-wt, with a peak at 559 nm from heme B and the broad absorption in the 585- to 600-nm region indicative of bound heme A (Fig. 7B). The spectra of reduced CtaA-H60L, CtaA-H123L, and CtaA-H123M were very similar to each other and showed the {alpha}-band absorption peak at 556 nm (Fig. 7B and spectra not shown). A peak at 556 nm correlated with the presence of mainly heme O bound to CtaA, and we therefore conclude that it is due to heme O. The observed 3-nm spectral difference between heme O and heme B bound to CtaA is congruent with the pyridine hemochrome spectrum of heme O, which is blue shifted 4 nm in comparison to that of heme B (33). The tight binding of heme O to the protein and the distinct spectra typical of low-spin cytochrome indicated that the overall structure of the mutant enzymes was not much disturbed compared to that of the wild-type protein.

Reduced CtaA-H278L and CtaA-H278M showed little absorption in the 500- to 700-nm region (spectra not shown), which agreed with the low heme content of these mutant variants.

The H216M variant of CtaA binds a heme A synthase reaction intermediate. CtaA-H216M showed features that were unique among the mutants analyzed. This CtaA variant has a decreased heme A synthase activity. Membranes of LMT20R/pH216M contained wild-type levels of CtaA protein (Fig. 4) but contained small amounts of cytochrome a (Fig. 3, trace G) and showed a fivefold reduction in cytochrome c oxidase activity compared to that of membranes from LMT20R/pCTHI10 (Table 3). The CtaA-H216M variant contained heme B, heme O, and a third heme compound which we identified as heme I based on the HPLC elution pattern (Fig. 6) and the light absorption spectrum in acidic acetonitrile with an absorption maximum at 394 nm. Hegg et al. (3) have isolated heme I from membranes of recombinant E. coli strains containing B. subtilis CtaA and identified it as monohydroxymethyl heme O, which is a proposed heme A synthase reaction intermediate (Fig. 8). The light absorption spectrum of the dithionite-reduced CtaA-H216M protein showed a broader {alpha}-band absorption peak compared to that of CtaA containing only heme B or heme O (Fig. 7). This indicates that heme B (peak at 559 nm) and heme O (peak at 556 nm), and possibly also heme I, contribute to the {alpha}-band absorption peak in the spectrum of dithionite-reduced CtaA-H216M.



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  		FIG. 8. Proposed three-step reaction for the heme O-to-heme A synthesis catalyzed by heme A synthase. Mutations in B. subtilis CtaA that have been used in this work and negatively affect reaction steps are indicated (see text for details); a solid line indicates a block and a dashed line indicates decreased activity of heme A synthase.


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DISCUSSION
 
Heme A synthase catalyzes the synthesis of heme A from heme O, which involves the conversion of a methyl side group on the heme O porphyrin ring into a formyl group (Fig. 8). We have in this work analyzed the role of four invariant histidine residues in heme A synthases by using the B. subtilis heme A synthase, CtaA, as a model enzyme. We first established a system for production and easy isolation of wild-type and mutant variants of CtaA in B. subtilis. To our knowledge, B. subtilis CtaA is the only heme A synthase that has been isolated in preparative amounts and analyzed in any detail (reference 24 and this work).

The functions of the four invariant histidine residues in heme A synthases, located at positions 60, 123, 216, and 278 in the B. subtilis CtaA protein sequence (Fig. 2), were analyzed by changing each residue individually into a leucine and a methionine by site-directed mutagenesis. Our results show that His-60, -123, and -216 are important for heme A synthase activity but are not required for binding of the enzyme substrate, heme O, or heme B, which might function as a prosthetic group in CtaA. The results also indicate that His-278, located in the most C-terminal transmembrane segment of CtaA (Fig. 2), is not critical for heme A synthase activity but seems to play a role in the tight binding of heme to the protein since the His-278 mutant variants CtaA-H278L and CtaA-H278M both contained little bound heme compared to the wild type.

The N- and C-terminal halves of CtaA, containing transmembrane segments I to IV and V to IIX, respectively, are homologous and might each constitute a four-helix bundle protein domain (25). Residues His-60 and His-216 have equivalent positions in the two halves (Fig. 2). Substitution of His-60 and His-216 with a leucine or a methionine affected the heme A synthase activity and heme content of CtaA in ways which are indicative of a role for these two histidine residues in heme coordination. A methionine at position 60 results in active enzyme with the same heme composition and light absorption spectrum as wild-type CtaA. In contrast, a leucine residue in position 60 of CtaA resulted in an inactive enzyme protein containing tightly bound heme O. Methionine, but not leucine, can function as an axial heme ligand and could substitute for a histidine residue in this respect. This can explain why the CtaA-H60M, but not the CtaA-H60L, enzyme is catalytically active, assuming that His-60 plays a role in heme coordination. CtaA protein with a leucine in position 216 could not be detected in membranes or be purified. Defective heme ligation could result in improper folding or decreased stability of the CtaA polypeptide in the membrane, which in turn could lead to degradation of the polypeptide. A methionine at position 216 in CtaA resulted in a stable enzyme protein with decreased heme A synthase activity. This CtaA variant accumulated bound heme I (monohydroxymethyl heme O), indicating a deficiency in step 2 of the proposed enzyme reaction (Fig. 8). The presence of heme O and heme I in purified mutant CtaA proteins supports the view that CtaA catalyzes the entire heme O-to-heme A synthesis reaction. A methionine residue instead of a histidine as the axial ligand to heme iron is expected to increase the midpoint redox potential of that heme. For example, a histidine-to-methionine substitution of an axial heme ligand in the cytochrome b subunit of B. subtilis succinate-quinone oxidoreductase increases the midpoint potential of that heme by more than 100 mV (14). Such a large change in redox potential is expected to have a significant effect on the reactivity of the heme and can explain the deficiency of the CtaA-H216M variant. It is not known if CtaA has one or two heme-binding sites and if axial ligand switching to heme iron occurs during the catalytic cycle, as has been observed, for example, in Thiosphaera pantotropha cytochrome cd1 nitrite reductase (31) and Nitrosomonas europaea cytochrome c peroxidase (23). We can therefore only speculate on whether His-60 and His-216 in CtaA would be ligands to the same heme or to two different heme groups.

Residue His-123 in CtaA seems to be crucial for step 1 in the proposed heme A synthase reaction (Fig. 8). Both CtaA-H123L and CtaA-H123M were found to be inactive but formed stable proteins containing heme O and heme B and showed spectra similar to that of the CtaA-H60L variant. We suggest that His-123 functions directly in catalysis.

In conclusion, we have established an experimental platform for functional analysis of heme A synthase in B. subtilis. Mutant variants of the enzyme have for the first time been purified and analyzed with respect to in vivo activity and heme content. Analysis of the mutant heme A synthase variants allowed dissection of the biosynthetic reaction performed by the enzyme and provided strong support for the proposed enzyme reaction pathway (Fig. 8).


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ACKNOWLEDGMENTS
 
We are grateful to Ingrid Stål for expert technical assistance.

This work was supported by grants 621-2001-3125 and 621-2004-2762 from the Swedish Science Research Council.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Cell & Organism Biology, Lund University, Sölvegatan 35, SE-223 62 Lund, Sweden. Phone: 46 (46) 2228622. Fax: 46 (46) 2224113. E-mail: Lars.Hederstedt{at}cob.lu.se. Back

{dagger} Present address: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway. Back


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Journal of Bacteriology, December 2005, p. 8361-8369, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8361-8369.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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