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Structure and metal centers
STRUCTURE AND METAL CENTERS
The Protein Formula of Beef Heart Cytochrome c Oxidase Gerhard Buse, Lothar Meinecke, and Bernhard Bruch Abteilung Physiologische Chemie, R WTH Aachen, West Germany
ABSTRACT Beef heart cytochrome c oxidase consist of 12 different polypeptides stoichiometrically arranged in respiratory complex IV. The functional 2 heme a, 2 copper monomer of this complex consist of 1793 amino acids; the exact M, is 202,787 Da. From 17 cystein residues, six are involved in the formation of three disulphide bonds. The theoretical heme a content of the enzyme is 9.86 nmol/mg protein. The theoretical iron and copper contents are 0.55 and 0.63 pg/mg protein, respectively.
INTRODUCTION
There has been long debate about the number of subunits constituting mitochondrial cytochrome c oxidase (EC 1.9.3. l), respiratory complex IV. Recently, however, evidence has accumulated that the number of protein components has been underestimated and is at least 9 in the yeast enzyme [l] and 12 to 13 in the enzyme complex prepared from mammalian mitochondria [2-4]. It has been argued that some of the smaller nuclear coded polypeptides are impurities copurifying with the enzyme complex in common preparations, and, since no function can be attributed to these proteins until now, they have not been considered as “subunits” of the enzyme [5, 61. On the one hand this argument is a kind of indefinite judgment that is not acceptable since the investigation of this enzyme is not complete. On the other hand the argument presently would pertain to all nuclear coded polypeptides of the oxidase complex and thus focuses to the now well-known fact that the established redox and H + -translocating functions of the enzyme originate from the mitochondrially coded proteins [7, 81. These are the true subunits of an enzymatic function that is of procaryotic origin. In eucaryotic cells we find a symbiotic adaptation of this function, different in phyla, in tissues, and probably in ontogenetic development, the biochemical expression of which is well known as respiratory “complex.” Address reprint requests to Dr. Gerhard Technische Hochschule, Aachen Klinikum Journal of Inorganic Biochemistry 0 1985 Elsevier Science Publishing 52 Vanderbilt
Physiologische Buse, Abteilung Pauwelsstrasse, D-5100 Aachen,
23, 149-153 Co., Inc.
Ave., New York, NY 10017
Chemie,
Bheinisch-Westfalische
West Germany.
(1985) 149 0162-0134/85/%3.30
150
G. Buse,
L. Meinecke,
and B. Bruch
METHODS l The stoichiometry of the components was investigated 1. by Edman degradation of the intact enzyme and HPLC-quantification of the resulting PTH-amino acids through several cycles. This automated procedure is known to work with > 95 % accuracy [lo]; 2. by hydrazinolysis [ 1 l] and carboxypeptidase digestion of the enzyme and determination of the C-terminal residues by amino acid analysis; 3. by summative calculation of the theoretical uv-absorption at 280 nm (tryptophan and tyrosine content) of all polypeptides and the enzyme, and comparison with the data for the intact enzyme and its polypeptides separated on Bio Gel P-100 columns in 3% sodium dodecyl sulfate. The cysteine status of the enzyme was investigated after labeling of all free sulfhydryl residues with an excess of 2-iodo acetic acid under denaturating conditions in 8 M urea/Tris HCl, pH 8.6. The enzyme was then reduced with either 2-mercaptoethanol or dithioerythreitol (DTE) and additional cysteine residues were reacted with the fluorescent label 5-N-iodoacetylethylendiaminenapthalene-sulfonic acid (1.5 J-AEDANS) or with the colored thiol specific reagents 4-N-dimethylamino-3,5-dinitrophenyl-malimide (Tuppy’s malimide), 1 bromoacetamino-4-nitro-2-hydroxybenzene (Koshland III), or 4-N-dimethylamino-azobenzene-4-N’-iodoacetamide (DABIA). These thiol specific reagents were also used for an additional detection of the free sulfhydryl groups under denaturating conditions in 8 M urea or 3 % SDS, pH 8.6 with prevention of reduction/oxidation in a nitrogen atmosphere. Separation and identification of the labeled polypeptides was done by SDSpolyacrylamide gel electrophoresis, Bio-Gel chromatography in 3% SDS or 10% acetic acid, and amino acid analysis as described elsewhere [9; footnote 11.
RESULTS The complex of the active bovine heart cytochrome c oxidase has by the vast majority of all investigators been found to have z 10 nMo1 heme a/mg protein, despite different ways of solubilization and purification [ 121. For the two heme a, two copper functional unit of the enzyme a protein M, close to 200,000 Da must result. This is clearly the case if the 12 polypeptide pattern which we have found from the protein chemical analysis [2] is taken as a basis. Table 1 lists all the protein components of beef heart cytochrome c oxidase, purified to 10 nMo1 heme a/mg protein with their exact A4, and the N- and C-terminal sequences. After completion of all primary structures of the enzyme complex we now were able to determine the stoichiometry of all protein constituents with the entire complex by nondissociating exact methods. The results consistently show that: there are 12 different polypeptides, no other polypeptides are detectable, there is exact 1: 1 stoichiometry with the exception of VIIIb (Ile), with exact stoichiometry ’ For all the sequences of the bovine heart cytochrome c oxidase polypeptides and the methods for their determination see the series of papers “Studies on Cytochrome c Oxidase ” in Hoppe-Seyler’s Zeitschrift fiir Physiologische Chemie; last issue: Ref. 9.
56993“ 26049 29918” 17153 12436 10668 9419 8480 10063 5441 4962 6243
0 From mtDNA sequence 1131.
VIIIC
VlIIa VW
VII
WC
I II III N V Vki VIb
Polypeptide
2S-H
2s-s
IS-H 2S-H; lS-S lS-H
lS-H 2S-H 2S-H
Cysteine status
Cyt. Cyt. Cyt. Cyt.
Cyt
Mit. Mit. Cyt. Cyt. Cyt. Cyt.
Mit.
Synthesis
TABLE 1. Protein Components of Bovine Heart Cytochrome c Oxidase
Formyl-Met-Phe-Ile-AsnFormyl-Met-Ala-Tyr-Pro(Met)Thr-Hi-GlnAla-His-Gly-SerSer-His-Gly-SerAla-Ser-Gly-GlyAla-Ser-Ala-AlaSer-Thr-Ala-LeuAcetyl-Ala-Glu-Asp-IleSer-His-Tyr-GluIle-Thr-Ala-LysPhe-Glu-Asn-Arg
N-terminal sequences
-Val-Asn-Leu-Lys -Ala-k-Met&w -Trp-Ttp-Gly-Ser -Gin-Ttp-Lys-Lys -Leu-AspLys-Val Gin-Leu-Ala-His -Gly-Tyr-Glu-Lys -Gin-Ser-Ala-Lys -Pro-Gly-Lys-Ile -Leu-Leu-Lys-Lys -Ser-!&-Ala-Ala -Ala-Ser-Lys-Lys
sequences
C-terminal
1
227
514
1
10 5 17 22 1.5 6 13 8 8 12 16 18 34 11 6 6 7 6 2 5
16 19 38 30 9 6 28 47 40 38 34 37 59 19 42 9 17 8 1 17
II
’ M, (Protein): 202787 Da.
Stoichiometry
CYs Trp
Arg
TY~ Phe LYs His
LeU
Asp Asn Thr Ser GlU Gln Pro Gly Ala Val Met Ile
I
1
261
4 6 24 19 8 7 12 21 14 16 11 14 31 11 24 3 17 5 2 12
III
TABLE 2. Amino Acid Composition
9 4 5 4 1 11 7 6 8 8 1 7 11 4 3 7 3 I 1 2
109
147
V
8 4 5 12 13 3 6 5 13 11 4 5 11 7 7 18 4 5 0 6
IV
and Stoichiometry
98
4 4 7 5 8 4 7 9 8 6 1 5 7 2 1 6 4 4 4 2
Via
~_ __
84 ._..~
2 4 7 5 2 0 7 9 7 2 0 2 8 2 6 3 7 7 1 3 ~_ ~_.
VII,
73 ~_.
I 4 11 3 4 2 5 4 8 8 1 7 0 0
1
3 1 2 4 4
VIC
of the Protein Components
_..~ 85
6 5 5 5 4 5 4 4 6 4 1 4 2 4 4 6 1 7 4 4
VU
~_
47
46
1 1 4 5 1 1 4 2 6 2 0 2 6 2 2 5 1 0 0 1
0
2 1 3 3 1 3 4 3 2 2 2 5 1 6 4 2 2 0 1
VIIIb
c Oxidase,
VIIIa
of Bovine Cytochrome
56
2 3 4 2 3 2 1 6 3 3 1 1 9 3 2 5 2 2 2 0
VIIIC
1793
66 59 123 121 72 48 97 127 133 109 75 101 194 72 113 85 67 60 17 54
Complex IV”
100.02
3.68 3.29 6.86 6.74 4.07 2.67 5.41 7.08 7.42 6.08 4.18 5.63 10.82 4.01 6.30 4.74 3.74 3.34 0.95 3.01
%
Protein Formula of Beef Heart Cytochrome
c Oxidase
153
2. The complex therefore consists of 13 proteins in the 2 heme, 2 copper functional unit. As shown in Table 2 the enzyme contains 17 cysteine residues. As results from the differential labeling assays, were only two cysteine residues react in polypeptide Via and all four cysteine residues in polypeptide VII, from these 17 residues 6 are involved in the formation of three disulfide bridges, one in polypeptide Via and two in polypeptide VII. With these determinations the data for the protein formula of beef heart cytochrome c oxidase are complete. The monomer is built up from altogether 1793 amino acids with an exact A4, of 202.787 Da. The theoretical heme a content then is 9.86 nMol/mg protein. The theoretical iron and copper contents are 0.55 and 0.627 pg/mg protein, respectively. This work was
by the
105,499-507 (1980). R. A. Capaldi, Biochim. Biophys. Acta 694, 291-306 (1982). M. Saraste, Trends Biochem. Sci. 8, 139-142 (1983). B. Ludwig, Biochim. Biophys. Acta 594, 177-189 (1980). G. C. M. Steffens, G. Buse, W. Oppliger, and B. Ludwig, Biochem. Biophys. Res. Commun. 116, 335-340 (1983). L. Meinecke, G. J. Steffens, and G. Buse, Hoppe-Seyler’s Z. Physiol. Chem. 365, 313-320 (1984). P. Edman and G. Begg, Eur. J. Biochem. 1, 80-91 (1967). V. Braun and W. A. Schrijder, Arch. Biochem. Biophys. 118, 241-252 (1967). W. S. Caughey, W. J. Wallace, J. A. Volpe, and S. Yoshikawa, in The Enzymes, P. D. Boyer, Ed., Academic, New York 1976, 3rd edn., Vol. 13c, pp. 299-344. S. Anderson, M. H. L. de Bruijn, A. R. Coulson, I. C. Eperon, F. Sanger, and I. G. Young, J. Mol. Biol. 156, 683-717 (1982). Received and accepted November 1984
The Protein Formula of Beef Heart Cytochrome c Oxidase Gerhard Buse, Lothar Meinecke, and Bernhard Bruch Abteilung Physiologische Chemie, R WTH Aachen, West Germany
ABSTRACT Beef heart cytochrome c oxidase consist of 12 different polypeptides stoichiometrically arranged in respiratory complex IV. The functional 2 heme a, 2 copper monomer of this complex consist of 1793 amino acids; the exact M, is 202,787 Da. From 17 cystein residues, six are involved in the formation of three disulphide bonds. The theoretical heme a content of the enzyme is 9.86 nmol/mg protein. The theoretical iron and copper contents are 0.55 and 0.63 pg/mg protein, respectively.
INTRODUCTION
There has been long debate about the number of subunits constituting mitochondrial cytochrome c oxidase (EC 1.9.3. l), respiratory complex IV. Recently, however, evidence has accumulated that the number of protein components has been underestimated and is at least 9 in the yeast enzyme [l] and 12 to 13 in the enzyme complex prepared from mammalian mitochondria [2-4]. It has been argued that some of the smaller nuclear coded polypeptides are impurities copurifying with the enzyme complex in common preparations, and, since no function can be attributed to these proteins until now, they have not been considered as “subunits” of the enzyme [5, 61. On the one hand this argument is a kind of indefinite judgment that is not acceptable since the investigation of this enzyme is not complete. On the other hand the argument presently would pertain to all nuclear coded polypeptides of the oxidase complex and thus focuses to the now well-known fact that the established redox and H + -translocating functions of the enzyme originate from the mitochondrially coded proteins [7, 81. These are the true subunits of an enzymatic function that is of procaryotic origin. In eucaryotic cells we find a symbiotic adaptation of this function, different in phyla, in tissues, and probably in ontogenetic development, the biochemical expression of which is well known as respiratory “complex.” Address reprint requests to Dr. Gerhard Technische Hochschule, Aachen Klinikum Journal of Inorganic Biochemistry 0 1985 Elsevier Science Publishing 52 Vanderbilt
Physiologische Buse, Abteilung Pauwelsstrasse, D-5100 Aachen,
23, 149-153 Co., Inc.
Ave., New York, NY 10017
Chemie,
Bheinisch-Westfalische
West Germany.
(1985) 149 0162-0134/85/%3.30
150
G. Buse,
L. Meinecke,
and B. Bruch
METHODS l The stoichiometry of the components was investigated 1. by Edman degradation of the intact enzyme and HPLC-quantification of the resulting PTH-amino acids through several cycles. This automated procedure is known to work with > 95 % accuracy [lo]; 2. by hydrazinolysis [ 1 l] and carboxypeptidase digestion of the enzyme and determination of the C-terminal residues by amino acid analysis; 3. by summative calculation of the theoretical uv-absorption at 280 nm (tryptophan and tyrosine content) of all polypeptides and the enzyme, and comparison with the data for the intact enzyme and its polypeptides separated on Bio Gel P-100 columns in 3% sodium dodecyl sulfate. The cysteine status of the enzyme was investigated after labeling of all free sulfhydryl residues with an excess of 2-iodo acetic acid under denaturating conditions in 8 M urea/Tris HCl, pH 8.6. The enzyme was then reduced with either 2-mercaptoethanol or dithioerythreitol (DTE) and additional cysteine residues were reacted with the fluorescent label 5-N-iodoacetylethylendiaminenapthalene-sulfonic acid (1.5 J-AEDANS) or with the colored thiol specific reagents 4-N-dimethylamino-3,5-dinitrophenyl-malimide (Tuppy’s malimide), 1 bromoacetamino-4-nitro-2-hydroxybenzene (Koshland III), or 4-N-dimethylamino-azobenzene-4-N’-iodoacetamide (DABIA). These thiol specific reagents were also used for an additional detection of the free sulfhydryl groups under denaturating conditions in 8 M urea or 3 % SDS, pH 8.6 with prevention of reduction/oxidation in a nitrogen atmosphere. Separation and identification of the labeled polypeptides was done by SDSpolyacrylamide gel electrophoresis, Bio-Gel chromatography in 3% SDS or 10% acetic acid, and amino acid analysis as described elsewhere [9; footnote 11.
RESULTS The complex of the active bovine heart cytochrome c oxidase has by the vast majority of all investigators been found to have z 10 nMo1 heme a/mg protein, despite different ways of solubilization and purification [ 121. For the two heme a, two copper functional unit of the enzyme a protein M, close to 200,000 Da must result. This is clearly the case if the 12 polypeptide pattern which we have found from the protein chemical analysis [2] is taken as a basis. Table 1 lists all the protein components of beef heart cytochrome c oxidase, purified to 10 nMo1 heme a/mg protein with their exact A4, and the N- and C-terminal sequences. After completion of all primary structures of the enzyme complex we now were able to determine the stoichiometry of all protein constituents with the entire complex by nondissociating exact methods. The results consistently show that: there are 12 different polypeptides, no other polypeptides are detectable, there is exact 1: 1 stoichiometry with the exception of VIIIb (Ile), with exact stoichiometry ’ For all the sequences of the bovine heart cytochrome c oxidase polypeptides and the methods for their determination see the series of papers “Studies on Cytochrome c Oxidase ” in Hoppe-Seyler’s Zeitschrift fiir Physiologische Chemie; last issue: Ref. 9.
56993“ 26049 29918” 17153 12436 10668 9419 8480 10063 5441 4962 6243
0 From mtDNA sequence 1131.
VIIIC
VlIIa VW
VII
WC
I II III N V Vki VIb
Polypeptide
2S-H
2s-s
IS-H 2S-H; lS-S lS-H
lS-H 2S-H 2S-H
Cysteine status
Cyt. Cyt. Cyt. Cyt.
Cyt
Mit. Mit. Cyt. Cyt. Cyt. Cyt.
Mit.
Synthesis
TABLE 1. Protein Components of Bovine Heart Cytochrome c Oxidase
Formyl-Met-Phe-Ile-AsnFormyl-Met-Ala-Tyr-Pro(Met)Thr-Hi-GlnAla-His-Gly-SerSer-His-Gly-SerAla-Ser-Gly-GlyAla-Ser-Ala-AlaSer-Thr-Ala-LeuAcetyl-Ala-Glu-Asp-IleSer-His-Tyr-GluIle-Thr-Ala-LysPhe-Glu-Asn-Arg
N-terminal sequences
-Val-Asn-Leu-Lys -Ala-k-Met&w -Trp-Ttp-Gly-Ser -Gin-Ttp-Lys-Lys -Leu-AspLys-Val Gin-Leu-Ala-His -Gly-Tyr-Glu-Lys -Gin-Ser-Ala-Lys -Pro-Gly-Lys-Ile -Leu-Leu-Lys-Lys -Ser-!&-Ala-Ala -Ala-Ser-Lys-Lys
sequences
C-terminal
1
227
514
1
10 5 17 22 1.5 6 13 8 8 12 16 18 34 11 6 6 7 6 2 5
16 19 38 30 9 6 28 47 40 38 34 37 59 19 42 9 17 8 1 17
II
’ M, (Protein): 202787 Da.
Stoichiometry
CYs Trp
Arg
TY~ Phe LYs His
LeU
Asp Asn Thr Ser GlU Gln Pro Gly Ala Val Met Ile
I
1
261
4 6 24 19 8 7 12 21 14 16 11 14 31 11 24 3 17 5 2 12
III
TABLE 2. Amino Acid Composition
9 4 5 4 1 11 7 6 8 8 1 7 11 4 3 7 3 I 1 2
109
147
V
8 4 5 12 13 3 6 5 13 11 4 5 11 7 7 18 4 5 0 6
IV
and Stoichiometry
98
4 4 7 5 8 4 7 9 8 6 1 5 7 2 1 6 4 4 4 2
Via
~_ __
84 ._..~
2 4 7 5 2 0 7 9 7 2 0 2 8 2 6 3 7 7 1 3 ~_ ~_.
VII,
73 ~_.
I 4 11 3 4 2 5 4 8 8 1 7 0 0
1
3 1 2 4 4
VIC
of the Protein Components
_..~ 85
6 5 5 5 4 5 4 4 6 4 1 4 2 4 4 6 1 7 4 4
VU
~_
47
46
1 1 4 5 1 1 4 2 6 2 0 2 6 2 2 5 1 0 0 1
0
2 1 3 3 1 3 4 3 2 2 2 5 1 6 4 2 2 0 1
VIIIb
c Oxidase,
VIIIa
of Bovine Cytochrome
56
2 3 4 2 3 2 1 6 3 3 1 1 9 3 2 5 2 2 2 0
VIIIC
1793
66 59 123 121 72 48 97 127 133 109 75 101 194 72 113 85 67 60 17 54
Complex IV”
100.02
3.68 3.29 6.86 6.74 4.07 2.67 5.41 7.08 7.42 6.08 4.18 5.63 10.82 4.01 6.30 4.74 3.74 3.34 0.95 3.01
%
Protein Formula of Beef Heart Cytochrome
c Oxidase
153
2. The complex therefore consists of 13 proteins in the 2 heme, 2 copper functional unit. As shown in Table 2 the enzyme contains 17 cysteine residues. As results from the differential labeling assays, were only two cysteine residues react in polypeptide Via and all four cysteine residues in polypeptide VII, from these 17 residues 6 are involved in the formation of three disulfide bridges, one in polypeptide Via and two in polypeptide VII. With these determinations the data for the protein formula of beef heart cytochrome c oxidase are complete. The monomer is built up from altogether 1793 amino acids with an exact A4, of 202.787 Da. The theoretical heme a content then is 9.86 nMol/mg protein. The theoretical iron and copper contents are 0.55 and 0.627 pg/mg protein, respectively. This work was
by the
105,499-507 (1980). R. A. Capaldi, Biochim. Biophys. Acta 694, 291-306 (1982). M. Saraste, Trends Biochem. Sci. 8, 139-142 (1983). B. Ludwig, Biochim. Biophys. Acta 594, 177-189 (1980). G. C. M. Steffens, G. Buse, W. Oppliger, and B. Ludwig, Biochem. Biophys. Res. Commun. 116, 335-340 (1983). L. Meinecke, G. J. Steffens, and G. Buse, Hoppe-Seyler’s Z. Physiol. Chem. 365, 313-320 (1984). P. Edman and G. Begg, Eur. J. Biochem. 1, 80-91 (1967). V. Braun and W. A. Schrijder, Arch. Biochem. Biophys. 118, 241-252 (1967). W. S. Caughey, W. J. Wallace, J. A. Volpe, and S. Yoshikawa, in The Enzymes, P. D. Boyer, Ed., Academic, New York 1976, 3rd edn., Vol. 13c, pp. 299-344. S. Anderson, M. H. L. de Bruijn, A. R. Coulson, I. C. Eperon, F. Sanger, and I. G. Young, J. Mol. Biol. 156, 683-717 (1982). Received and accepted November 1984