BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1491-1437
Vol. 189, No. 3, 1992 December 30, 1992
HEME 0 BIOSYNTHESIS IN ESCHERZCHZA COLI: THE CYOE GENE IN THE CYTOCHROME BO OPERON ENCODES A PROTOHEME IX FARNESYLTRANSFERASEg Keitarou Saiki, Tatsushi Mogi and Yasuhiro Anraku* Department of Biology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Received
November
14,
1992
SUMMARY: The cytochrome bo complex of Escherichia coli is encoded by the cyoABCDE operon and functions as a redox-coupled proton pump. In this study, we have constructed eight cyoE deletion mutants and found that all the mutants were nonfunctional. Spectroscopic and heme analyses of the mutant oxidases revealed that the mutations specifically substituted protoheme IX for heme 0 present in the high-spin heme binding site. We found also that the overexpression of the cyoE gene in a cyo operon deletion strain resulted in a conversion of protoheme IX to heme 0. Since the CyoE protein contains the putative allylic polyprenyldiphosphate binding domain, we concluded that the cyoE gene encodes a novel 0 1992 Academl‘ enzyme, protoheme IX famesyltransferase, essential for heme 0 biosynthesis. Press.Inc.
The cytochrome bo complex, a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli, functions as a redox-coupled proton pump and belongs to the heme-copper respiratory oxidase superfamily (1). The cyoABCDE operon encoding the complex has been cloned and sequenced (2,3). The oxidase contains low-spin heme, high-spin heme and CUB in subunit I (4,5) and both hemes have been considered to be protoheme IX for a long time (6). Wikstrijm and collaborators (7,8) recently found that it contains heme 0 having a 17-carbon hydroxyethylfarnesyl side chain at position 2, as in heme A of cytochrome c oxidases, and a methyl group at position 8, as in protoheme IX (heme B), of the tetrapyrrole macrocycle. Since the famesylated hemes are exclusively found in the terminal oxidases, heme 0 and heme A are believed to be essential for the dioxygen reduction chemistry coupled to proton pumping,
#This is Paper III in the series “Structure-function studies on the E. coli cytochrome bo complex.” *To whom correspondence should be addressed.
1491
0006-29 I X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproductim in any form rrsrrvrd.
Vol.
189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
therefore, the aerobic respiration. However, the enzymatic mechanism for farnesylation of hemes remains unknown. Thus, heme 0 biosynthesis in E. cofi has become an urgent interest in bioenergetics. We have initiated the structure-function studies on the CyoE protein via sitedirected mutagenesis and identified that the cyoE mutations specifically affect the high-spin heme. We report here evidence that the cyoE gene is involved in heme 0 biosynthesis and suggest that it encodes a novel enzyme, protoheme IX famesyltransferase. MATERIALS
AND METHODS
Constructions of the cyoE deletion mutants --- AEl was made by blunt-end ligation at the EcoRV andNru1 sites and lacks about 80% of the CyoE (Ile-64 to Trp-296) and the cyo terminator (15). AE2 to AE7 were constructed via oligonucleotide-directed site-specific mutagenesis (4). AE2 was designed to eliminate the entire cyoE gene while AE2’ was made similarly but lacks the cyo terminator. AE3 to AE6 were designed to introduce small deletions in the cytoplasmic loops (“Arg-62 to Arg-74” in loop II-III, “Val-154 to Phe-l69” and “Gin-189 to Pro-197” in loop IV-V, and “Lys-253 to Lys-263” in loop VI-VII, respectively). AE7 lacks a carboxy-terminal half of the transmembrane region VII and a protruding end (Val-281 to Trp296). The mutants cyo operons whose nucleotides sequences have been thoroughly confumed by sequencing were introduced into the single copy expression vector pMFO1 (4). Complementationtest --- The in vivo catalytic activity of the mutant oxidases were examined by the aerobic growth of a terminal oxidase deficient strain ST2592 (Acyo Acyd recA) harboring the pMFO1 derivatives using minimal-glucose and -glycerol plates (4). Characterizationsof the mutant oxidases --- Cytoplasmic membrane vesicles were prepared from strain ST4676 (Acyo) harboring the pMFO1 derivatives and the cytochrome bo complex was purified from the membranes as described previously (4). Spectroscopic and immunochemical analysis, determinations of copper and heme contents, and assay of ubiquinol oxidase activity using ubiquinol-1 (a generous gift from Dr. S. Ohsono, Eizai Co. Ltd., Tsukuba) were carried out as described (4). Analysis of heme species--- Hemes were extracted from the purified oxidases or cytoplasmic membranes by acid acetone and analyzed by reverse phase HPLC as described (7). The solvent was 95% ethanol/acetic acid/water (70:17:7, vol/vol), and the flow rate was 0.5 ml/mm Elution profile was monitored by average absorbance at 396-402 nm. Expression of the cyoE gene by a tat promoter-directedexpressionvector --- The cyoE gene was subcloned into p’ITQl8 to give pTTQl8-cyoE (15) and expressed in strain ST4676 (Acyo) at the log phase of the growth by addition of 1 m M isopropyl B-D-thio-galactopyranoside RESULTS AND DISCUSSION The cyoE homologues have been found not only in operon structures for bacterial austype cytochrome c oxidases in Bacillus subtilis (9), thermophilic Bacillus PS3 (10) and Pamcoccus denitrijkans
(1 l), but also in yeast as the nuclear gene COXIO (12). The deletions
of the yeast COXIO gene (12) and the ctaB gene in P. denitrijicans (13) am known to cause a deficiency of subunit I, the redox center in the oxidase complex. The cyoE gene product (296 residues) has been predicted to consist of seven transmembrane segments (Fig. 1) (3,14). Eighteen out of the 23 invariant residues are present in the putative cytoplasmic loops II-III and IV-V, suggesting that these loops are likely to be the catalytic domains in the CyoE protein. In order to explore the functional role of the CyoE homologues in the heme-copper terminal 1492
Vol.
189,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
00 0.3
.: Ii 0 :
0.cooti
COMMUNICATIONS
296
Periplasm 1.-----------
--~
: :0
Cytoplasm
Fig. A topological model for the CyoE protein of the cytochrome ho complex showing locations of conserved amino acid residues. The model is based on the Kyte-Doolittle hydropathy profile (3) and gene fusion experiments (14). The putative membrane-spanning regions are indicated by rectangles.
oxidases, we have constructed and characterized eight cyoE deletion mutants and found from the aerobic complementation test that all the mutant oxidases are nonfunctional (Table 1). It indicates that the cyoE gene product is essential for the catalytic activity or the in vivo assembly
Table 1, Characterization of the cyoE mutant oxidases. The cytochrome o content was estimated from CO-reduced minus reduced difference spectra of cytoplasmic membranes at room temperature. The low-spin heme content was evaluated by a peak at 563.5 nm in second order finite spectra of dithionite-reduced minus air-oxidized difference spectra of cytoplasmic membranes at 77 K. Strains carrying mini-F plasmid pMFO1 and pHNF-2 (15) were used as the wild-type control and a vector control (control), respectively.
Mutant
Aerobic growth
Wild-type control
++
AEl (164-W296) AE2 (Ml-W296) AE2'(Ml-W296) AE3 (R62-R74) AE4 (V154-F169) AE5 (Q189-P197) AE6 (K253-K263) AE7 (V281-W296)
-
(nmol/mg cyt
0
0.39 0.05a O.llb
o.14b 0.08b o.lS=
0.19 0.19 o.17b 0.19’
protein) CU
Low spin heme
0.33
+++
0.01
-
0.19 0.18 0.14 0.21 0.18 0.13 0.21 0.26
+ ++ ++ ++ ++ ++ ++
Subunit +++
++ +++ + +++ +++ ++ +++ +++
a: CO-binding by the cytochrome bd complex. b: Accompanied by l-3 nm red-shifts of the Soret peak : the CO-bound, reduced state. 1493
I
Vol. 189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the oxidase complex. Western immunoblotting analysis indicated that the defects were not associated with a loss of subunits I (Table 1) and II (data not shown). Here, we should stress that small deletions in the cytoplasmic loops, the deletions AE3 to AE6 (Fig. l), were as effective as a complete loss of the CyoE, AE2, supporting the idea that the cytoplasmic loops are the functional domain of the CyoE. Spectroscopic analysis of the metal centers of the mutant oxidases in cytoplasmic membranes revealed that all the deletions specifically reduced the CO-binding activity and caused 1 to 3 nm red-shifts of the Soret peaks of the high-spin heme (Table 1). Furthermore, we have purified the AE2 mutant oxidase and found that it contains all the five subunits (75,33.5,28,20.5
and 12 kDa polypeptides) present in the wild-type oxidase (data
not shown). This eliminates the possible role of the CyoE as the nucleation core of the oxidase complex. Spectroscopic analysis confirmed that the AE2 deletion specifically altered the highspin heme. In the purified AE2 mutant oxidase, the CO-binding activity was reduced to one sixth of the wild-type level and the Soret peak of the high-spin heme was red-shifted as in the cytoplasmic membranes, whereas the low-spin heme and CUB were not affected by the mutation (Fig. 2, Table 2). We found that the a-peak of the pyridine ferrohemochrome from the AE2 mutant oxidase was shifted from 554.5 nm of the wild-type to 556.5 nm which is identical to that of protoheme IX from bovine hemoglobin. Reverse phase HPLC analysis confirmed that the wild-type oxidase contains one mole each of protoheme IX and heme 0 while the AE2 mutant oxidase contains two protoheme IX molecules (Fig. 3ab, Table 2). These results indicate that the cyoE deletions caused the substitution of protoheme IX for heme 0 present in the high-spin heme binding site. Thus, the cyoE mutation converts the oxidase from the heme BO-type to the
b
1 ,I/ 400
450
500 500
550
600
Wavelength (nm) F&J.
Spectroscopic analysis of the purified wild-type (WT) and AE2 oxidase. a. CO-reduced
minus reduced difference spectra at room temperature. b. Second order finite spectra of dithionite-reduced minus air-oxidized difference spectra at 77 K. Spectra were recorded at protein concentrations of 0.2 and 1.0 mg protein/ml of 50 m M Tris-HCl (pH 7.4) containing 0.1% sucrose monolaurate, respectively. 1494
Vol.
189,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table 2. Effects of a total loss of the cyoE gene product on the contents of the metal centers in subunit1 of the purified cytochrome bo complex. Heme contents are expressed as a sum of protoheme IX and heme 0.
(nmol/mg cyt Wild AE2
type
2.3 0.4
0
Quinol
protein) cu
Heme
2.8 2.1
6.5 4.1
oxidase
(pm01 QlHa/min.mg
protein)
54.1
Vol. 189, No. 3, 1992 December 30, 1992
HEME 0 BIOSYNTHESIS IN ESCHERZCHZA COLI: THE CYOE GENE IN THE CYTOCHROME BO OPERON ENCODES A PROTOHEME IX FARNESYLTRANSFERASEg Keitarou Saiki, Tatsushi Mogi and Yasuhiro Anraku* Department of Biology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Received
November
14,
1992
SUMMARY: The cytochrome bo complex of Escherichia coli is encoded by the cyoABCDE operon and functions as a redox-coupled proton pump. In this study, we have constructed eight cyoE deletion mutants and found that all the mutants were nonfunctional. Spectroscopic and heme analyses of the mutant oxidases revealed that the mutations specifically substituted protoheme IX for heme 0 present in the high-spin heme binding site. We found also that the overexpression of the cyoE gene in a cyo operon deletion strain resulted in a conversion of protoheme IX to heme 0. Since the CyoE protein contains the putative allylic polyprenyldiphosphate binding domain, we concluded that the cyoE gene encodes a novel 0 1992 Academl‘ enzyme, protoheme IX famesyltransferase, essential for heme 0 biosynthesis. Press.Inc.
The cytochrome bo complex, a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli, functions as a redox-coupled proton pump and belongs to the heme-copper respiratory oxidase superfamily (1). The cyoABCDE operon encoding the complex has been cloned and sequenced (2,3). The oxidase contains low-spin heme, high-spin heme and CUB in subunit I (4,5) and both hemes have been considered to be protoheme IX for a long time (6). Wikstrijm and collaborators (7,8) recently found that it contains heme 0 having a 17-carbon hydroxyethylfarnesyl side chain at position 2, as in heme A of cytochrome c oxidases, and a methyl group at position 8, as in protoheme IX (heme B), of the tetrapyrrole macrocycle. Since the famesylated hemes are exclusively found in the terminal oxidases, heme 0 and heme A are believed to be essential for the dioxygen reduction chemistry coupled to proton pumping,
#This is Paper III in the series “Structure-function studies on the E. coli cytochrome bo complex.” *To whom correspondence should be addressed.
1491
0006-29 I X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproductim in any form rrsrrvrd.
Vol.
189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
therefore, the aerobic respiration. However, the enzymatic mechanism for farnesylation of hemes remains unknown. Thus, heme 0 biosynthesis in E. cofi has become an urgent interest in bioenergetics. We have initiated the structure-function studies on the CyoE protein via sitedirected mutagenesis and identified that the cyoE mutations specifically affect the high-spin heme. We report here evidence that the cyoE gene is involved in heme 0 biosynthesis and suggest that it encodes a novel enzyme, protoheme IX famesyltransferase. MATERIALS
AND METHODS
Constructions of the cyoE deletion mutants --- AEl was made by blunt-end ligation at the EcoRV andNru1 sites and lacks about 80% of the CyoE (Ile-64 to Trp-296) and the cyo terminator (15). AE2 to AE7 were constructed via oligonucleotide-directed site-specific mutagenesis (4). AE2 was designed to eliminate the entire cyoE gene while AE2’ was made similarly but lacks the cyo terminator. AE3 to AE6 were designed to introduce small deletions in the cytoplasmic loops (“Arg-62 to Arg-74” in loop II-III, “Val-154 to Phe-l69” and “Gin-189 to Pro-197” in loop IV-V, and “Lys-253 to Lys-263” in loop VI-VII, respectively). AE7 lacks a carboxy-terminal half of the transmembrane region VII and a protruding end (Val-281 to Trp296). The mutants cyo operons whose nucleotides sequences have been thoroughly confumed by sequencing were introduced into the single copy expression vector pMFO1 (4). Complementationtest --- The in vivo catalytic activity of the mutant oxidases were examined by the aerobic growth of a terminal oxidase deficient strain ST2592 (Acyo Acyd recA) harboring the pMFO1 derivatives using minimal-glucose and -glycerol plates (4). Characterizationsof the mutant oxidases --- Cytoplasmic membrane vesicles were prepared from strain ST4676 (Acyo) harboring the pMFO1 derivatives and the cytochrome bo complex was purified from the membranes as described previously (4). Spectroscopic and immunochemical analysis, determinations of copper and heme contents, and assay of ubiquinol oxidase activity using ubiquinol-1 (a generous gift from Dr. S. Ohsono, Eizai Co. Ltd., Tsukuba) were carried out as described (4). Analysis of heme species--- Hemes were extracted from the purified oxidases or cytoplasmic membranes by acid acetone and analyzed by reverse phase HPLC as described (7). The solvent was 95% ethanol/acetic acid/water (70:17:7, vol/vol), and the flow rate was 0.5 ml/mm Elution profile was monitored by average absorbance at 396-402 nm. Expression of the cyoE gene by a tat promoter-directedexpressionvector --- The cyoE gene was subcloned into p’ITQl8 to give pTTQl8-cyoE (15) and expressed in strain ST4676 (Acyo) at the log phase of the growth by addition of 1 m M isopropyl B-D-thio-galactopyranoside RESULTS AND DISCUSSION The cyoE homologues have been found not only in operon structures for bacterial austype cytochrome c oxidases in Bacillus subtilis (9), thermophilic Bacillus PS3 (10) and Pamcoccus denitrijkans
(1 l), but also in yeast as the nuclear gene COXIO (12). The deletions
of the yeast COXIO gene (12) and the ctaB gene in P. denitrijicans (13) am known to cause a deficiency of subunit I, the redox center in the oxidase complex. The cyoE gene product (296 residues) has been predicted to consist of seven transmembrane segments (Fig. 1) (3,14). Eighteen out of the 23 invariant residues are present in the putative cytoplasmic loops II-III and IV-V, suggesting that these loops are likely to be the catalytic domains in the CyoE protein. In order to explore the functional role of the CyoE homologues in the heme-copper terminal 1492
Vol.
189,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
00 0.3
.: Ii 0 :
0.cooti
COMMUNICATIONS
296
Periplasm 1.-----------
--~
: :0
Cytoplasm
Fig. A topological model for the CyoE protein of the cytochrome ho complex showing locations of conserved amino acid residues. The model is based on the Kyte-Doolittle hydropathy profile (3) and gene fusion experiments (14). The putative membrane-spanning regions are indicated by rectangles.
oxidases, we have constructed and characterized eight cyoE deletion mutants and found from the aerobic complementation test that all the mutant oxidases are nonfunctional (Table 1). It indicates that the cyoE gene product is essential for the catalytic activity or the in vivo assembly
Table 1, Characterization of the cyoE mutant oxidases. The cytochrome o content was estimated from CO-reduced minus reduced difference spectra of cytoplasmic membranes at room temperature. The low-spin heme content was evaluated by a peak at 563.5 nm in second order finite spectra of dithionite-reduced minus air-oxidized difference spectra of cytoplasmic membranes at 77 K. Strains carrying mini-F plasmid pMFO1 and pHNF-2 (15) were used as the wild-type control and a vector control (control), respectively.
Mutant
Aerobic growth
Wild-type control
++
AEl (164-W296) AE2 (Ml-W296) AE2'(Ml-W296) AE3 (R62-R74) AE4 (V154-F169) AE5 (Q189-P197) AE6 (K253-K263) AE7 (V281-W296)
-
(nmol/mg cyt
0
0.39 0.05a O.llb
o.14b 0.08b o.lS=
0.19 0.19 o.17b 0.19’
protein) CU
Low spin heme
0.33
+++
0.01
-
0.19 0.18 0.14 0.21 0.18 0.13 0.21 0.26
+ ++ ++ ++ ++ ++ ++
Subunit +++
++ +++ + +++ +++ ++ +++ +++
a: CO-binding by the cytochrome bd complex. b: Accompanied by l-3 nm red-shifts of the Soret peak : the CO-bound, reduced state. 1493
I
Vol. 189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the oxidase complex. Western immunoblotting analysis indicated that the defects were not associated with a loss of subunits I (Table 1) and II (data not shown). Here, we should stress that small deletions in the cytoplasmic loops, the deletions AE3 to AE6 (Fig. l), were as effective as a complete loss of the CyoE, AE2, supporting the idea that the cytoplasmic loops are the functional domain of the CyoE. Spectroscopic analysis of the metal centers of the mutant oxidases in cytoplasmic membranes revealed that all the deletions specifically reduced the CO-binding activity and caused 1 to 3 nm red-shifts of the Soret peaks of the high-spin heme (Table 1). Furthermore, we have purified the AE2 mutant oxidase and found that it contains all the five subunits (75,33.5,28,20.5
and 12 kDa polypeptides) present in the wild-type oxidase (data
not shown). This eliminates the possible role of the CyoE as the nucleation core of the oxidase complex. Spectroscopic analysis confirmed that the AE2 deletion specifically altered the highspin heme. In the purified AE2 mutant oxidase, the CO-binding activity was reduced to one sixth of the wild-type level and the Soret peak of the high-spin heme was red-shifted as in the cytoplasmic membranes, whereas the low-spin heme and CUB were not affected by the mutation (Fig. 2, Table 2). We found that the a-peak of the pyridine ferrohemochrome from the AE2 mutant oxidase was shifted from 554.5 nm of the wild-type to 556.5 nm which is identical to that of protoheme IX from bovine hemoglobin. Reverse phase HPLC analysis confirmed that the wild-type oxidase contains one mole each of protoheme IX and heme 0 while the AE2 mutant oxidase contains two protoheme IX molecules (Fig. 3ab, Table 2). These results indicate that the cyoE deletions caused the substitution of protoheme IX for heme 0 present in the high-spin heme binding site. Thus, the cyoE mutation converts the oxidase from the heme BO-type to the
b
1 ,I/ 400
450
500 500
550
600
Wavelength (nm) F&J.
Spectroscopic analysis of the purified wild-type (WT) and AE2 oxidase. a. CO-reduced
minus reduced difference spectra at room temperature. b. Second order finite spectra of dithionite-reduced minus air-oxidized difference spectra at 77 K. Spectra were recorded at protein concentrations of 0.2 and 1.0 mg protein/ml of 50 m M Tris-HCl (pH 7.4) containing 0.1% sucrose monolaurate, respectively. 1494
Vol.
189,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table 2. Effects of a total loss of the cyoE gene product on the contents of the metal centers in subunit1 of the purified cytochrome bo complex. Heme contents are expressed as a sum of protoheme IX and heme 0.
(nmol/mg cyt Wild AE2
type
2.3 0.4
0
Quinol
protein) cu
Heme
2.8 2.1
6.5 4.1
oxidase
(pm01 QlHa/min.mg
protein)
54.1
