Bioehimiea et Biophysica Acta, 393 (1975) 48-54 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37036 T H E S U B U N I T S T R U C T U R E OF P S E U D O M O N A S C Y T O C H R O M E OX1DASE
TAPANI KURONEN*, MATTI SARASTE and NILS ELLFOLK Department of Biochemistry, University of Helsinki, SF-O0170 Helsinki (Finland) (Received November 18th, 1974)
SUMMARY
Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63 000 and, according to the iron determination, contains two heroes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68 ~ of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (Sz0,w ~ 4 S) from 18 to 9 2 ~ . At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63 000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M NazSO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaCI solutions is partly explained by preferential hydratation of the protein.
INTRODUCTION
Pseudomonas cytochrome c:oxygen oxidoreductase (EC 1.9.3.2), has been classified as cytochrome cdl [1]. The electron donors of the enzyme are cytochrome c-551 (P. aeruginosa) and azurin (P. aeruginosa), with oxygen and nitrite both functioning as electron acceptors [2]. A discrepancy with earlier molecular weight determinations [3, 4] was resolved in two recent independent studies [5, 6], in both of which a molecular weight of approx. 120 000 was obtained. We were able to show that the molecule was a dimer which could be dissociated into subunits by succinylation and dodecylsulphate treatment [5]. In independent measurements by dodecylsulphate/acrylamide gel electrophoresis the molecular weight of the subunit was found to be 58 000-63 000 [5, 6]. These values are consistent with the amino acid composition data [4]. According to our iron determination and the iron content given by Nagata et al. [4] the subunit of * Present address: Central Public Health Laboratory SF-00280 Helsinki (Finland).
49 cytochrome oxidase contains two heroes. This contradicts the results of Gudat et al. who found only one heme per subunit [6]. In the present study we have made a new determination of the iron content of the cytochrome oxidase. In addition the influence of succinylation, pH and salts on the dissociation of the dimer into subunits has been studied. MATERIALS AND METHODS The purification procedure, the activity assays, the concentration measurement of the cytochrome oxidase and methods used in the ultracentrifuge experiments have all been described previously [5]. Commercial guanidine hydrochloride [7] and urea [8] were purified before use.
Succinylation [1,4-t4C]Succinic anhydride, 50 #Ci/mg (New England Nuclear Corp.) was mixed with unlabelled succinic anhydride (Fluka) and dissolved in dry acetone to yield a concentration of 100 mg/ml and 50/zCi/ml. This solution was added dropwise to the 1 ~ cytochrome oxidase solution at 20 °C. The pH of the reaction mixture was maintained between 6 and 8 by the addition of 1 M NaOH using a Radiometer autotitrator. During the succinylation procedure aliquots were taken and dialyzed exhaustively against phosphate buffer (pH 7.0, I = 0.1) at 4 °C. The radioactivity of the dialyzed preparations was determined with a Wallac 81 000 liquid scintillation counter. Protein concentrations were estimated by measuring the absorbance at 280 nm. The absorption coefficient of the preparation, E i1% = 18.5, showed no cm measurable change during the procedure.
Partial specific volume The partial specific volume of cytochrome oxidase was determined by the mechanical oscillator technique [9] using a DMA-10 densimeter (A. Paar, Graz, Austria). The temperature (20 °C) was kept constant within & 0.01 °C. The samples were dialyzed for 30 h at 4 °C.
Iron assay The iron content was measured with a Techtron AA-4 atomic absorption spectrophotometer equipped with a graphite oven. The wavelength used was 242.8 nm. The concentration range was 50-100 ng Fe/ml. Mohr's salt (p.a. Merck) was used as an iron standard. The cytochrome oxidase was dialyzed exhaustively against 0.2 NH4HCO3 before the analyses. RESULTS Because of the discrepancy in the iron content of the cyt0chrome oxidase, a new technique was used to determine the iron. The value of 0.154~ obtained confirmed our earlier results (0.166~o), i.e. two iron atoms per subunit.
Succinylation Cytochrome oxidase was treated with increasing amounts of [~4C]succinic
5O Meniscus
Fig. 1. Sedimentation velocity patterns of succinylated cytochrome oxidase numbered in order of increasing modification. Figures are drawn from the photographs taken 65 rain after reaching the operating speed of 59 780 rev./min. Protein concentrations of the fractions from S-1 to S-6 are 8.2, 7.0, 6.4, 5.2, 4,9 and 3.4 g/1. The sedimentation coefficients of the peaks are given in the figure.
anhydride under neutral conditions. Six preparations were obtained and numbered f r o m S-1 to S-6 in order of increasing succinylation. The excess of succinic anhydride relative to the lysine content of the cytochrome oxidase [4] varied from 3:1 in S-I to 20:1 in S-6. The sedimentation patterns of the succinylated enzyme preparations are shown in Fig. 1. Three sedimentation peaks were observed, at 6.7-6.9 S, 4.0-4.5 S and 2-2.3 S. These will be referred to as components 7 S, 4 S and 2 S. With increasing succinylation (S-1-S-4) increasing amounts of the 4 S component were observed (Fig. 2). The formation of the 2 S components occurred only in preparations with high succinylation (S-5, S-6). In dodecylsulphate/acrylamide gel electrophoresis [10] all the fractions S-1-S-6 moved as a single band with molecular weight of approx. 65 000. The enzymatic activity and the relative amount of the 7 S component decreased in parallel with increasing modification (Fig. 2). There was no difference between the loss of the cytochrome oxidase and nitrite reductase activity.
The effect of pH The effect of pH on the dissociation of the enzyme molecule and the activity of the cytochrome oxidase was followed in low ionic strength buffers in the p H range
51
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TAPANI KURONEN*, MATTI SARASTE and NILS ELLFOLK Department of Biochemistry, University of Helsinki, SF-O0170 Helsinki (Finland) (Received November 18th, 1974)
SUMMARY
Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63 000 and, according to the iron determination, contains two heroes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68 ~ of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (Sz0,w ~ 4 S) from 18 to 9 2 ~ . At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63 000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M NazSO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaCI solutions is partly explained by preferential hydratation of the protein.
INTRODUCTION
Pseudomonas cytochrome c:oxygen oxidoreductase (EC 1.9.3.2), has been classified as cytochrome cdl [1]. The electron donors of the enzyme are cytochrome c-551 (P. aeruginosa) and azurin (P. aeruginosa), with oxygen and nitrite both functioning as electron acceptors [2]. A discrepancy with earlier molecular weight determinations [3, 4] was resolved in two recent independent studies [5, 6], in both of which a molecular weight of approx. 120 000 was obtained. We were able to show that the molecule was a dimer which could be dissociated into subunits by succinylation and dodecylsulphate treatment [5]. In independent measurements by dodecylsulphate/acrylamide gel electrophoresis the molecular weight of the subunit was found to be 58 000-63 000 [5, 6]. These values are consistent with the amino acid composition data [4]. According to our iron determination and the iron content given by Nagata et al. [4] the subunit of * Present address: Central Public Health Laboratory SF-00280 Helsinki (Finland).
49 cytochrome oxidase contains two heroes. This contradicts the results of Gudat et al. who found only one heme per subunit [6]. In the present study we have made a new determination of the iron content of the cytochrome oxidase. In addition the influence of succinylation, pH and salts on the dissociation of the dimer into subunits has been studied. MATERIALS AND METHODS The purification procedure, the activity assays, the concentration measurement of the cytochrome oxidase and methods used in the ultracentrifuge experiments have all been described previously [5]. Commercial guanidine hydrochloride [7] and urea [8] were purified before use.
Succinylation [1,4-t4C]Succinic anhydride, 50 #Ci/mg (New England Nuclear Corp.) was mixed with unlabelled succinic anhydride (Fluka) and dissolved in dry acetone to yield a concentration of 100 mg/ml and 50/zCi/ml. This solution was added dropwise to the 1 ~ cytochrome oxidase solution at 20 °C. The pH of the reaction mixture was maintained between 6 and 8 by the addition of 1 M NaOH using a Radiometer autotitrator. During the succinylation procedure aliquots were taken and dialyzed exhaustively against phosphate buffer (pH 7.0, I = 0.1) at 4 °C. The radioactivity of the dialyzed preparations was determined with a Wallac 81 000 liquid scintillation counter. Protein concentrations were estimated by measuring the absorbance at 280 nm. The absorption coefficient of the preparation, E i1% = 18.5, showed no cm measurable change during the procedure.
Partial specific volume The partial specific volume of cytochrome oxidase was determined by the mechanical oscillator technique [9] using a DMA-10 densimeter (A. Paar, Graz, Austria). The temperature (20 °C) was kept constant within & 0.01 °C. The samples were dialyzed for 30 h at 4 °C.
Iron assay The iron content was measured with a Techtron AA-4 atomic absorption spectrophotometer equipped with a graphite oven. The wavelength used was 242.8 nm. The concentration range was 50-100 ng Fe/ml. Mohr's salt (p.a. Merck) was used as an iron standard. The cytochrome oxidase was dialyzed exhaustively against 0.2 NH4HCO3 before the analyses. RESULTS Because of the discrepancy in the iron content of the cyt0chrome oxidase, a new technique was used to determine the iron. The value of 0.154~ obtained confirmed our earlier results (0.166~o), i.e. two iron atoms per subunit.
Succinylation Cytochrome oxidase was treated with increasing amounts of [~4C]succinic
5O Meniscus
Fig. 1. Sedimentation velocity patterns of succinylated cytochrome oxidase numbered in order of increasing modification. Figures are drawn from the photographs taken 65 rain after reaching the operating speed of 59 780 rev./min. Protein concentrations of the fractions from S-1 to S-6 are 8.2, 7.0, 6.4, 5.2, 4,9 and 3.4 g/1. The sedimentation coefficients of the peaks are given in the figure.
anhydride under neutral conditions. Six preparations were obtained and numbered f r o m S-1 to S-6 in order of increasing succinylation. The excess of succinic anhydride relative to the lysine content of the cytochrome oxidase [4] varied from 3:1 in S-I to 20:1 in S-6. The sedimentation patterns of the succinylated enzyme preparations are shown in Fig. 1. Three sedimentation peaks were observed, at 6.7-6.9 S, 4.0-4.5 S and 2-2.3 S. These will be referred to as components 7 S, 4 S and 2 S. With increasing succinylation (S-1-S-4) increasing amounts of the 4 S component were observed (Fig. 2). The formation of the 2 S components occurred only in preparations with high succinylation (S-5, S-6). In dodecylsulphate/acrylamide gel electrophoresis [10] all the fractions S-1-S-6 moved as a single band with molecular weight of approx. 65 000. The enzymatic activity and the relative amount of the 7 S component decreased in parallel with increasing modification (Fig. 2). There was no difference between the loss of the cytochrome oxidase and nitrite reductase activity.
The effect of pH The effect of pH on the dissociation of the enzyme molecule and the activity of the cytochrome oxidase was followed in low ionic strength buffers in the p H range
51
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° i 3.e
0.~
QJ
o.6 ° v 0J
o.4 ~ g12
IF 0.4
c O
