FEMS Microbiology Letters 100 (1992) 91-100 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
91
FEMSLE 80031
Molecular biology of Bacillus subtilis cytochromes Claes von W a c h e n f e l d t a n d L a r s H e d e r s t e d t Department of Microbiology, Uniuersity of Lund, Lund, Sweden Received 12 June 1992 Accepted 15 June 1992
Key words: Bacillus subtilis; Cytochrome; Heme protein; Bacterial respiration
1. S U M M A R Y
Bacillus subtilis cells must have cytochromes for growth and can synthesize cytochromes of a-, b-, c-, d-, and o-types. After a long lag, our knowledge of the structure, genetics and specific role for these cytochromes is now growing exponentially as the result of recent research. This progress is reviewed here and includes, for example, the discovery of two different cytochrome a systems and genes required for their biogenesis.
2. I N T R O D U C T I O N Our knowledge at the molecular level of respiratory components in the strictly aerobic, sporeforming, Gram-positive model-organism, Bacillus subtilis has rapidly expanded during the last few years. All the cytochromes of this bacterium appear to be membrane-bound and an unanticipated complexity in cytochrome composition has
Correspondence to: L. Hederstedt, Department of Microbiology, University of Lund, S61vegatan 21, S-223 62 Lund, Sweden.
been revealed from biochemical and genetic studies. This recent progress, together with the fact that relatively few molecular data are available on respiratory components in Gram-positive bacteria in general, make it timely to review the information obtained so far. In this short paper we survey, from a molecular view, the cytochromes in Bacillus subtilis 168 strains. Cytochromes are defined as heme-proteins in which the heme prosthetic group undergoes oxid a t i o n / reduction as part of the function of the protein. The classification of cytochromes into different types is based on the chemical structure of the heme and how the heme is bound to the cytochrome polypeptide; a-type cytochromes contain heme A, b-type cytochromes have heme B (protoheme IX), c-type cytochromes harbour protoheme IX covalently bound to one or in most cases two cysteine residues, d-type cytochromes contain a chlorin-type of heme, o-type cytochromes may generally contain the recently discovered heme O. The structure of heine O differs from protoheme IX in that the vinyl group on carbon 3 is replaced by a hydroxyethylfarnesyl chain. Heme A differs from heme O in that the methyl group on carbon 18 of the tetrapyrrole ring is oxidised to a formyl group [1].
92 The cytochrome composition of B. subtilis membranes isolated from vegetative cells varies depending on the strain, the growth conditions and growth stage [cf. 2,3]. This variability is illustrated in Fig. 1 by the light absorption spectra of cytoplasmic membranes from cells grown in different liquid media. Eleven different cytochromes or cytochrome-containing enzyme complexes have so far been identified in B. subtilis (Table 1). This diversity in cytochrome composition is not obvious from spectra (Fig. 1) because several of the cytochromes show essentially overlapping absorption or are present in low amounts. A genetic approach combined with biochemical analyses is now being pursued in several laboratories in order to try to elucidate the B. subtilis cytochrome arsenal and to analyse the structure, biogenesis and physiological function in the cell of the respective cytochrome. In B. subtilis more than 500 genetic loci have been m a p p e d on the chromosome which is comprised of 4165 kbp [4,5]. The structural genes for five cytochromes have been cloned, sequenced and located on the circular, 360 °, chromosomal map (Table 1). Cytochrome genes are not clustered on the chromosome or linked to genes required for protoheme IX synthesis ( h e m A X C D B L at 244 ° [6] and h e m E H Y at 94 ° (Hansson, M. and Hederstedt, L., manuscript in preparation)) or menaquinone-7 synthesis ( m e n -
B C D at 273 °) [7]. Menaquinone-7, which is the only quinone found in B. subtilis membranes [8],
serves as electron carrier between different redoxproteins such as dehydrogenases and cytochromes [cf. 9,10]. In the following sections we present the state of knowledge regarding the genetics, function and structure of the hitherto identified cytochromes listed in Table 1.
3. C Y T O C H R O M E b-558 OF C O M P L E X II 3.1. Function o f cytochrome b-558
Cytochrome b-558 is the most abundant cytochrome in membranes isolated from B. subtilis grown in a complex medium such as NSMP or in minimal-succinate medium (Fig. 1) [11,12]. This relatively well characterised b-type cytochrome is a subunit of the succinate : menaquinone-7 reductase (Complex II) and is functionally an integral component of both the Krebs' cycle and the respiratory chain. Complex II is comprised of three subunits; a 65 kDa flavoprotein subunit (Fp) containing a covalently bound FAD, a 28 kDa ironsulphur protein subunit (Ip) containing a [2Fe-2S], a [3Fe-4S], and a [4Fe-4S] iron-sulphur centre, and a 23 kDa cytochrome b-558 subunit containing two protoheme IX molecules [12]. The cytochrome subunit spans the cytoplasmic membrane and anchors the Fp and Ip subunits tightly
Table I Bacillus subtUis membrane-bound cytochromes
Cytoehrome
cyt c-550 (13 kDa)
Function Cytochrome oxidase Quinol oxidase Subunit of succinate : menaquinone oxidoreductase (Complex II) Unknown (required for expression of a-type cytochromes) Unknown (not essential)
cyt c-554 (29 kDa?) and cyt b-562
Possible subunits of a bc1-1ikecomplex
Unknown
24,27,29
cyt c (52 kDa) cyt c (22 kDa) cyt d
Unknown Unknown Oxidase?
Unknown Unknown Unknown
24,27 24,27 27
cyt o
Oxidase
Unknown
29,43
cyt caa 3 cyt aa 3 cyt b-558/sdh cyt b-558/cta
ctaCDEF (127°) qoxABCD (330°) sdhC (252°)
Genes (location)
Reference 25,27 37 12,18
ctaA and ctaB
27,50
(127°) cccA (223°)
24
93 to the cytoplasmic side of the m e m b r a n e [11]. U p o n oxidation of succinate to fumarate, two electrons are transferred via F A D , iron-sulphur centres and probably heme as intermediate electron carriers, to m e n a q u i n o n e - 7 in the m e m b r a n e lipid bilayer [12].
3.2. Properties and structure of cytochrome b-558 Spectroscopic and t h e r m o d y n a m i c properties of the two hemes in cytochrome b-558 are sum-
I
I
t
I
I
400
450
500
1
I
I~ 550
I
I
I
600
650
Wavelength (nm)
700
marised in Table 2. D a t a obtained using cryogenic electron p a r a m a g n e t i c resonance ( E P R ) and near-infrared magnetic circular dichroism ( N I R M C D ) in conjunction, strongly suggest bis-histidine ligation of both heme groups and a near perpendicular orientation of the imidazole-planes at each heine [13]. Two different models for the topography of the 202 amino acid residue cytochrome b-558 polypeptide in the m e m b r a n e have been proposed. O n e model suggests five t r a n s m e m b r a n e a-helical segments [14], the other model only four [15]. The five t r a n s m e m b r a n e segment model which places the N-terminus in the cytoplasm and the C-terminus on the outside of the cytoplasmic m e m b r a n e has the strongest experimental support as recently discussed [16]. Cytochrome b-558 contains six His residues. Results from sitespecific m u t a t i o n analysis ([14], H~igerh~ill, C. et al., unpublished data) in combination with sequence comparisons to the analogous cytochrome b of the WolineUa succinogenes fumarate reductase complex [17] strongly suggest that His-28, His-70, H i s - l l 3 and His-155 of B. subtilis cyt o c h r o m e b-558 are the axial ligands to heme. These four His residues are located in different postulated t r a n s m e m b r a n e segments (five segm e n t model). Which pair of His residues that ligates the high and low potential heme, respectively, is not known.
Fig. 1. Light absorption spectra (dithionite reduced minus oxidised) of B. subtilis membranes. The strain 168 (trpC2) obtained from the Bacillus Genetic Stock Center, OH (catalogue No. 1A1) was grown at 37°C in 1 1 of medium in 5-1 baffled E-flasks (200 rpm) and the cells were harvested when the culture reached the end of exponential growth phase. The bacteria were grown in the following media: A, minimal medium with 0.5% glutamate; B, minimal medium with 0.5% glycerol; C, minimal medium with 0.5% succinate; D, minimal medium with 0.5% glucose; E, nutrient broth sporulation medium with phosphate (NSMP); F, NSMP with 0.5% glucose. The Spizizen minimal medium and NSMP are described [11]. The protein concentration was 4 mg ml 1 in 20 mM MOPS/NaC1 buffer pH 7.4 and the spectra were recorded at room temperature in 10-mm path-length cuvenes. Different gains were used to record spectra in the 400-500 nm and 500-700 nm regions; the vertical bars indicate a difference in absorption of 0.1. The vertical dashed line is at 560 nm.
94 Table 2 Properties of the two heroes in isolated B. subtilis succinate : menaquinone oxidoreductase (Complex II) Succinate reducible " Midpoint potential (at pH 7.4) EPR signal (gmax) Light absorption spectrum b (Amaxof u-band): at room temp at 77 K
High potential heme Yes + 65 mV 3.68
Low potential heme No - 93 mV 3.42
558 nm 555 nm
558 nm 553, 558 nm c
~ Under steady state conditions. b Reduced state. ~"Split absorption peak. Data from [12].
3.3. Genetics of complex II The cytochrome b-558, Fp and Ip polypeptides are encoded by the sdhC, sdhA and sdhB genes, respectively [18,19]. These genes, located at 252 ° on the chromosome are transcribed into a 3.4-kb sdhCAB m R N A from a sigma-A dependent promoter [20]. Regulation of sdhCAB gene expression occurs predominantly at the m R N A level [21]. Addition of glucose to a complex growth medium (NSMP, see Fig. 1) results in decreased transcription from the sdh promoter and an approx. 6-fold lower steady state cellular concentration of sdh m R N A and Complex II protein in the m e m b r a n e compared to that in cells grown without added glucose. There is also post-transcriptional control [22,23]. The physical half-life of sdhCAB m R N A is influenced by whether the m R N A is translated and by the growth phase of the ceil, i.e. the half-life is 2.6 rain in exponentially growing cells compared to about 0.4 min in early stationary growth phase. B. subtilis mutants defective in expression of Complex II and with mutated structural genes have been isolated after both ' r a n d o m ' and site specific mutagenesis and characterised [11, 14, 16 and references therein].
4. C-TYPE C Y T O C H R O M E S
4.1. Sequence information In spite of the wealth of information on structure, function and evolutionary relationships of cytochromes c from Gram-negative bacteria and
eukaryotic cells, little is known about this group of cytochromes in Gram-positive bacteria. There are at present only four complete primary structures of Gram-positive cytochromes c available: B. subtilis cytochrome c-550 [24], Bacillus PS3 cytochrome c-551 (Sone, N. et al., unpublished data), the heme C containing subunit II of the cytochrome caa 3 oxidase from B. subtilis [25], and the thermophilic bacterium Bacillus PS3 [26], respectively.
4.2. Number and cellular location of cytochromes c Heme-proteins can be specifically labelled by growing cells in the presence of radioactive 5aminolevulinic acid, the first committed intermediate in heme biosynthesis [11,24,27]. When heme-proteins are denatured at 100°C in the presence of reducing agents and SDS, and then subjected to SDS-polyacrylamide gel electrophoresis, only covalently bound heme (heme C) remains attached to protein and can be detected by autoradiography. This method is considerably more sensitive than the heine staining procedure using tetramethylbenzidine [28]. Using this radioactive heme labelling technique up to five cytochrome c polypeptides with molecular masses of 16, 22, 29, 36 and 52 kDa can be detected in wild-type B. subtilis membranes [24,27]. All these polypeptides are found in the cytoplasmic m e m b r a n e and can be released only by using detergents [24]. Thus, all c-type cytochromes of B. subtilis behave as integral membrane proteins. Among the labelled polypeptides
95
the 16 kDa protein corresponds to cytochrome c-550 and the 36 kDa protein has been identified as subunit II of the cytochrome caa 3 oxidase [27]. The functions and properties of the c-type cytochromes corresponding to the 22, 29 and 52 kDa polypeptides have not been established. De Vrij et al. [29] described a cytochrome c-554 from B. subtilis strain W23 with a molecular mass of about 30 kDa that may correspond to the 29 kDa polypeptide. The role of cytochrome c-554 is not known, but it has been suggested to be a subunit of a bcl-like complex. The isolated bc 1 complex from Bacillus PS3 contains a 29 kDa cytochrome c I [30]. 4.3. Cytochrome c-550
Cytochrome c-550 is the smallest but one of the most abundant c-type cytochromes in B. subtilis [24,27]. Molecular and spectroscopic properties of cytochrome c-550 are presented in Table 3. The gene encoding cytochrome c-550, cccA located at 223 ° on the genetic map, has been isolated and sequenced [24]. As for many other bacterial c-type cytochromes [31] the specific function of B. subtilis cytochrome c-550 remains unknown. A mutant deleted of the entire cccA gene shows no apparent phenotypic defect [24]. The primary structure of cytochrome c-550 as deduced from the nucleotide sequence contains the conserved heme C binding motif, -C-X-Y-CH-, in the middle of the polypeptide (amino acid Table 3 Properties of bolocytochrome c-550 Amino acid residues a 120 Calculated mass ~ 13380 Da Estimated mass b 13000--16000 Da Calculated p l ~ 5.3 Net charge a -2 Light absorption spectra (hmax, reduced): at room temp. 415, 521,550 nm at 77 K 413, 519, 548 nm H e m e axial ligands E P R (gmax) Midpoint potential (Era. 7)
His-64, Met-99 3.4 + 178 _+6 mV
a From the deduced amino acid sequence. b Based upon SDS-polyacrylamide gel electrophoresis. Data from [24] and von Wachenfeldt, C. and Hederstedt, L., manuscripl in preparation.
A
B
~ C O O H
91
FEMSLE 80031
Molecular biology of Bacillus subtilis cytochromes Claes von W a c h e n f e l d t a n d L a r s H e d e r s t e d t Department of Microbiology, Uniuersity of Lund, Lund, Sweden Received 12 June 1992 Accepted 15 June 1992
Key words: Bacillus subtilis; Cytochrome; Heme protein; Bacterial respiration
1. S U M M A R Y
Bacillus subtilis cells must have cytochromes for growth and can synthesize cytochromes of a-, b-, c-, d-, and o-types. After a long lag, our knowledge of the structure, genetics and specific role for these cytochromes is now growing exponentially as the result of recent research. This progress is reviewed here and includes, for example, the discovery of two different cytochrome a systems and genes required for their biogenesis.
2. I N T R O D U C T I O N Our knowledge at the molecular level of respiratory components in the strictly aerobic, sporeforming, Gram-positive model-organism, Bacillus subtilis has rapidly expanded during the last few years. All the cytochromes of this bacterium appear to be membrane-bound and an unanticipated complexity in cytochrome composition has
Correspondence to: L. Hederstedt, Department of Microbiology, University of Lund, S61vegatan 21, S-223 62 Lund, Sweden.
been revealed from biochemical and genetic studies. This recent progress, together with the fact that relatively few molecular data are available on respiratory components in Gram-positive bacteria in general, make it timely to review the information obtained so far. In this short paper we survey, from a molecular view, the cytochromes in Bacillus subtilis 168 strains. Cytochromes are defined as heme-proteins in which the heme prosthetic group undergoes oxid a t i o n / reduction as part of the function of the protein. The classification of cytochromes into different types is based on the chemical structure of the heme and how the heme is bound to the cytochrome polypeptide; a-type cytochromes contain heme A, b-type cytochromes have heme B (protoheme IX), c-type cytochromes harbour protoheme IX covalently bound to one or in most cases two cysteine residues, d-type cytochromes contain a chlorin-type of heme, o-type cytochromes may generally contain the recently discovered heme O. The structure of heine O differs from protoheme IX in that the vinyl group on carbon 3 is replaced by a hydroxyethylfarnesyl chain. Heme A differs from heme O in that the methyl group on carbon 18 of the tetrapyrrole ring is oxidised to a formyl group [1].
92 The cytochrome composition of B. subtilis membranes isolated from vegetative cells varies depending on the strain, the growth conditions and growth stage [cf. 2,3]. This variability is illustrated in Fig. 1 by the light absorption spectra of cytoplasmic membranes from cells grown in different liquid media. Eleven different cytochromes or cytochrome-containing enzyme complexes have so far been identified in B. subtilis (Table 1). This diversity in cytochrome composition is not obvious from spectra (Fig. 1) because several of the cytochromes show essentially overlapping absorption or are present in low amounts. A genetic approach combined with biochemical analyses is now being pursued in several laboratories in order to try to elucidate the B. subtilis cytochrome arsenal and to analyse the structure, biogenesis and physiological function in the cell of the respective cytochrome. In B. subtilis more than 500 genetic loci have been m a p p e d on the chromosome which is comprised of 4165 kbp [4,5]. The structural genes for five cytochromes have been cloned, sequenced and located on the circular, 360 °, chromosomal map (Table 1). Cytochrome genes are not clustered on the chromosome or linked to genes required for protoheme IX synthesis ( h e m A X C D B L at 244 ° [6] and h e m E H Y at 94 ° (Hansson, M. and Hederstedt, L., manuscript in preparation)) or menaquinone-7 synthesis ( m e n -
B C D at 273 °) [7]. Menaquinone-7, which is the only quinone found in B. subtilis membranes [8],
serves as electron carrier between different redoxproteins such as dehydrogenases and cytochromes [cf. 9,10]. In the following sections we present the state of knowledge regarding the genetics, function and structure of the hitherto identified cytochromes listed in Table 1.
3. C Y T O C H R O M E b-558 OF C O M P L E X II 3.1. Function o f cytochrome b-558
Cytochrome b-558 is the most abundant cytochrome in membranes isolated from B. subtilis grown in a complex medium such as NSMP or in minimal-succinate medium (Fig. 1) [11,12]. This relatively well characterised b-type cytochrome is a subunit of the succinate : menaquinone-7 reductase (Complex II) and is functionally an integral component of both the Krebs' cycle and the respiratory chain. Complex II is comprised of three subunits; a 65 kDa flavoprotein subunit (Fp) containing a covalently bound FAD, a 28 kDa ironsulphur protein subunit (Ip) containing a [2Fe-2S], a [3Fe-4S], and a [4Fe-4S] iron-sulphur centre, and a 23 kDa cytochrome b-558 subunit containing two protoheme IX molecules [12]. The cytochrome subunit spans the cytoplasmic membrane and anchors the Fp and Ip subunits tightly
Table I Bacillus subtUis membrane-bound cytochromes
Cytoehrome
cyt c-550 (13 kDa)
Function Cytochrome oxidase Quinol oxidase Subunit of succinate : menaquinone oxidoreductase (Complex II) Unknown (required for expression of a-type cytochromes) Unknown (not essential)
cyt c-554 (29 kDa?) and cyt b-562
Possible subunits of a bc1-1ikecomplex
Unknown
24,27,29
cyt c (52 kDa) cyt c (22 kDa) cyt d
Unknown Unknown Oxidase?
Unknown Unknown Unknown
24,27 24,27 27
cyt o
Oxidase
Unknown
29,43
cyt caa 3 cyt aa 3 cyt b-558/sdh cyt b-558/cta
ctaCDEF (127°) qoxABCD (330°) sdhC (252°)
Genes (location)
Reference 25,27 37 12,18
ctaA and ctaB
27,50
(127°) cccA (223°)
24
93 to the cytoplasmic side of the m e m b r a n e [11]. U p o n oxidation of succinate to fumarate, two electrons are transferred via F A D , iron-sulphur centres and probably heme as intermediate electron carriers, to m e n a q u i n o n e - 7 in the m e m b r a n e lipid bilayer [12].
3.2. Properties and structure of cytochrome b-558 Spectroscopic and t h e r m o d y n a m i c properties of the two hemes in cytochrome b-558 are sum-
I
I
t
I
I
400
450
500
1
I
I~ 550
I
I
I
600
650
Wavelength (nm)
700
marised in Table 2. D a t a obtained using cryogenic electron p a r a m a g n e t i c resonance ( E P R ) and near-infrared magnetic circular dichroism ( N I R M C D ) in conjunction, strongly suggest bis-histidine ligation of both heme groups and a near perpendicular orientation of the imidazole-planes at each heine [13]. Two different models for the topography of the 202 amino acid residue cytochrome b-558 polypeptide in the m e m b r a n e have been proposed. O n e model suggests five t r a n s m e m b r a n e a-helical segments [14], the other model only four [15]. The five t r a n s m e m b r a n e segment model which places the N-terminus in the cytoplasm and the C-terminus on the outside of the cytoplasmic m e m b r a n e has the strongest experimental support as recently discussed [16]. Cytochrome b-558 contains six His residues. Results from sitespecific m u t a t i o n analysis ([14], H~igerh~ill, C. et al., unpublished data) in combination with sequence comparisons to the analogous cytochrome b of the WolineUa succinogenes fumarate reductase complex [17] strongly suggest that His-28, His-70, H i s - l l 3 and His-155 of B. subtilis cyt o c h r o m e b-558 are the axial ligands to heme. These four His residues are located in different postulated t r a n s m e m b r a n e segments (five segm e n t model). Which pair of His residues that ligates the high and low potential heme, respectively, is not known.
Fig. 1. Light absorption spectra (dithionite reduced minus oxidised) of B. subtilis membranes. The strain 168 (trpC2) obtained from the Bacillus Genetic Stock Center, OH (catalogue No. 1A1) was grown at 37°C in 1 1 of medium in 5-1 baffled E-flasks (200 rpm) and the cells were harvested when the culture reached the end of exponential growth phase. The bacteria were grown in the following media: A, minimal medium with 0.5% glutamate; B, minimal medium with 0.5% glycerol; C, minimal medium with 0.5% succinate; D, minimal medium with 0.5% glucose; E, nutrient broth sporulation medium with phosphate (NSMP); F, NSMP with 0.5% glucose. The Spizizen minimal medium and NSMP are described [11]. The protein concentration was 4 mg ml 1 in 20 mM MOPS/NaC1 buffer pH 7.4 and the spectra were recorded at room temperature in 10-mm path-length cuvenes. Different gains were used to record spectra in the 400-500 nm and 500-700 nm regions; the vertical bars indicate a difference in absorption of 0.1. The vertical dashed line is at 560 nm.
94 Table 2 Properties of the two heroes in isolated B. subtilis succinate : menaquinone oxidoreductase (Complex II) Succinate reducible " Midpoint potential (at pH 7.4) EPR signal (gmax) Light absorption spectrum b (Amaxof u-band): at room temp at 77 K
High potential heme Yes + 65 mV 3.68
Low potential heme No - 93 mV 3.42
558 nm 555 nm
558 nm 553, 558 nm c
~ Under steady state conditions. b Reduced state. ~"Split absorption peak. Data from [12].
3.3. Genetics of complex II The cytochrome b-558, Fp and Ip polypeptides are encoded by the sdhC, sdhA and sdhB genes, respectively [18,19]. These genes, located at 252 ° on the chromosome are transcribed into a 3.4-kb sdhCAB m R N A from a sigma-A dependent promoter [20]. Regulation of sdhCAB gene expression occurs predominantly at the m R N A level [21]. Addition of glucose to a complex growth medium (NSMP, see Fig. 1) results in decreased transcription from the sdh promoter and an approx. 6-fold lower steady state cellular concentration of sdh m R N A and Complex II protein in the m e m b r a n e compared to that in cells grown without added glucose. There is also post-transcriptional control [22,23]. The physical half-life of sdhCAB m R N A is influenced by whether the m R N A is translated and by the growth phase of the ceil, i.e. the half-life is 2.6 rain in exponentially growing cells compared to about 0.4 min in early stationary growth phase. B. subtilis mutants defective in expression of Complex II and with mutated structural genes have been isolated after both ' r a n d o m ' and site specific mutagenesis and characterised [11, 14, 16 and references therein].
4. C-TYPE C Y T O C H R O M E S
4.1. Sequence information In spite of the wealth of information on structure, function and evolutionary relationships of cytochromes c from Gram-negative bacteria and
eukaryotic cells, little is known about this group of cytochromes in Gram-positive bacteria. There are at present only four complete primary structures of Gram-positive cytochromes c available: B. subtilis cytochrome c-550 [24], Bacillus PS3 cytochrome c-551 (Sone, N. et al., unpublished data), the heme C containing subunit II of the cytochrome caa 3 oxidase from B. subtilis [25], and the thermophilic bacterium Bacillus PS3 [26], respectively.
4.2. Number and cellular location of cytochromes c Heme-proteins can be specifically labelled by growing cells in the presence of radioactive 5aminolevulinic acid, the first committed intermediate in heme biosynthesis [11,24,27]. When heme-proteins are denatured at 100°C in the presence of reducing agents and SDS, and then subjected to SDS-polyacrylamide gel electrophoresis, only covalently bound heme (heme C) remains attached to protein and can be detected by autoradiography. This method is considerably more sensitive than the heine staining procedure using tetramethylbenzidine [28]. Using this radioactive heme labelling technique up to five cytochrome c polypeptides with molecular masses of 16, 22, 29, 36 and 52 kDa can be detected in wild-type B. subtilis membranes [24,27]. All these polypeptides are found in the cytoplasmic m e m b r a n e and can be released only by using detergents [24]. Thus, all c-type cytochromes of B. subtilis behave as integral membrane proteins. Among the labelled polypeptides
95
the 16 kDa protein corresponds to cytochrome c-550 and the 36 kDa protein has been identified as subunit II of the cytochrome caa 3 oxidase [27]. The functions and properties of the c-type cytochromes corresponding to the 22, 29 and 52 kDa polypeptides have not been established. De Vrij et al. [29] described a cytochrome c-554 from B. subtilis strain W23 with a molecular mass of about 30 kDa that may correspond to the 29 kDa polypeptide. The role of cytochrome c-554 is not known, but it has been suggested to be a subunit of a bcl-like complex. The isolated bc 1 complex from Bacillus PS3 contains a 29 kDa cytochrome c I [30]. 4.3. Cytochrome c-550
Cytochrome c-550 is the smallest but one of the most abundant c-type cytochromes in B. subtilis [24,27]. Molecular and spectroscopic properties of cytochrome c-550 are presented in Table 3. The gene encoding cytochrome c-550, cccA located at 223 ° on the genetic map, has been isolated and sequenced [24]. As for many other bacterial c-type cytochromes [31] the specific function of B. subtilis cytochrome c-550 remains unknown. A mutant deleted of the entire cccA gene shows no apparent phenotypic defect [24]. The primary structure of cytochrome c-550 as deduced from the nucleotide sequence contains the conserved heme C binding motif, -C-X-Y-CH-, in the middle of the polypeptide (amino acid Table 3 Properties of bolocytochrome c-550 Amino acid residues a 120 Calculated mass ~ 13380 Da Estimated mass b 13000--16000 Da Calculated p l ~ 5.3 Net charge a -2 Light absorption spectra (hmax, reduced): at room temp. 415, 521,550 nm at 77 K 413, 519, 548 nm H e m e axial ligands E P R (gmax) Midpoint potential (Era. 7)
His-64, Met-99 3.4 + 178 _+6 mV
a From the deduced amino acid sequence. b Based upon SDS-polyacrylamide gel electrophoresis. Data from [24] and von Wachenfeldt, C. and Hederstedt, L., manuscripl in preparation.
A
B
~ C O O H
