JOURNAL OF BACTERIOLOGY, Jan. 1990, p. 465-468 0021-9193/90/010465-04$02.00/0 Copyright C) 1990, American Society for Microbiology
Vol. 172, No. 1
NOTES Purification and Properties of FerredoxinNAP, a Component of Naphthalene Dioxygenase from Pseudomonas sp. Strain NCIB 9816 BILLY E. HAIGLERt* AND DAVID T. GIBSONt Center for Applied Microb ology and Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received 19 May 1989/Accepted 2 October 1989
One of the three components of the naphthalene dioxygenase occurring in induced cells of Pseudomonas sp. strain NCIB 9816 has been purified to homogeneity. The protein contained 2 g-atoms each of iron and acid-labile sulfur and had an apparent molecular weight of 13,600. The evidence indicates that it is a ferredoxin-type protein that functions as an intermediate electron transfer protein in naphthalene dioxygenase activity.
Naphthalene dioxygenase is a multicomponent enzyme system which catalyzes the oxidation of naphthalene to cis-(lR,2S)-dihydroxy-1,2-dihydronaphthalene (4, 5). Naphthalene dioxygenase consists of three enzymes previously designated A, B, and C (5). Purification and characterization of component B showed that it was an iron-sulfur protein, designated 'SPNAP, that functioned as the terminal oxygenase (4). ISPNAP catalyzes the insertion of dioxygen into the aromatic ring of naphthalene (4). The ability of component A to transfer electrons from NADH to cytochrome c indicated that it was an NADH-oxidoreductase (4, 5). The activity of component A in the naphthalene dioxygenase and cytochrome c reductase assays was enhanced in the presence of flavin adenine dinucleotide (FAD) or flavin mononucleotide, with FAD being the preferred flavin cofactor (5, 9). Component A was shown to be an iron-sulfur flavoprotein which acts as the initial electron acceptor to shuttle electons from NADH to the terminal oxygenase (4, 5, 9). The function of component C was not clear, but it appeared to be an intermediate electron carrier in the enzyme system (4, 5). It was necessary to purify each of these proteins to establish their roles in naphthalene dioxygenation. Here we report the purification and characterization of ferredoxinNAP, previously designated component C. The results indicate that ferredoxinNAP is an iron-sulfur protein similar to the ferredoxin that functions in the toluene dioxygenase system (ferredoxinTOL) (16) and the ferredoxin component of the benzene dioxygenase system (2, 3). In another report, we show that ferredoxinNAP reductase, previously designated component A, can transfer electrons from NADH to ferredoxinNAP (9), thus supporting the role of ferredoxinNAp as an intermediate electron carrier in the naphthalene dioxygenase system. Pseudomonas sp. strain NCIB 9816 was grown and maintained as described previously (9). Lysis of the cells, protein and analytical determinations, and separation of ferre*
TABLE 1. Purification of ferredoxinNApa Purification step
Total Activity Sp act Purification protein (U) (U/mg) (fold) (mg)
Blue Sepharose 4,880 52,400 10.7 DEAE-cellulose 595 35.7 21,280 5.6 13,246 2,365 Sephadex G-75 DEAE-cellulose 3.2 10,800 3,375 a Details of the
purification procedure
Recovery (%)
100 41 25 21
3 221 315
are described in the text.
FerredoxinNAp-dependent naphthalene dioxygenase activity was measured in the presence of saturating levels of ferredoxinNAp reductase and ISPNAP. One unit of activity is defined as the amount of ferredoxinNAp required to oxidize 1.0 nmol of naphthalene per min.
doxinNAp and ISPNAP from NADH-ferredoxinNAP reductase by Blue Sepharose CL-6B gel chromatography were done as described before (9). Except where noted, all media, chemicals, and enzymes were those used in previous investigations (9, 16). Iron, amino acid, and acid-labile sulfur determinations were performed as previously described (9). FerredoxinNAP was located in column eluates by its ability to TABLE 2. Amino acid composition of ferredoxinNAP Amino acid
Lysine Arginine Threoninea Glycine Half-cystine" Methioninec Leucine
Phenylalanine Aspartate + asparagine
Corresponding author.
No. of residues/mol of enzyme
8 4 8 9 4 3 14 3 12
Amino acid
Histidine Serinea Proline Alanine Valine Isoleucine
Tyrosine Tryptophan Glutamate +
No. of residues/mol of enzyme
2 5 4
10 11 7 3 NDd 15
glutamine
a Determined by extrapolation to zero time of hydrolysis. b Determined after 24 h of hydrolysis in the presence of dimethyl sulfoxide
t Present address: Air Force Engineering and Services Center, Tyndall Air Force Base, FL 32403. t Present address: Department of Microbiology, College of Medicine, University of Iowa, Iowa City, IA 52242.
(15). ' d
465
Determined as methionine sulfone. ND, Not determined.
466
J. BACTERIOL.
NOTES
2.0
1.5
0.1
0.5
0.0
550
450
350
250
650
WAVELENGTH (nm) FIG. 1. Absorption spectrum of oxidized ferredoxinNAP. The vertical line indicates where the absorbance scale was changed.
catalyze naphthalene dioxygenase activity in the presence of
ferredoxinNAp reductase, ISPNAP, FAD, and NADH. Protein purification was carried out at 4°C. Fractions containing 'SPNAP and ferredoxinNAp activity eluted from the Blue Sepharose CL-6B column in the void volume and a portion of the buffer wash (9). The fractions containing ferredoxinNAp activity from two such column purifications (representing 100 g of cells [wet weight]) were pooled, centrifuged to remove particulate matter, and loaded on a DEAE-cellulose column (4 by 12 cm). The column was washed with 1 liter of TEG buffer (50 mM Tris hydrochloride buffer [pH 7.5] containing 10% [vol/vol] ethanol, 10% [vol/ vol] glycerol, and 0.5 mM dithiothreitol), and ISPNAP and ferredoxinNAP were separated with a continuous KCI gradient (0.0 to 0.4 M KCl) in 1 liter of TEG buffer. Fractions containing ISPNAP activity were pooled, concentrated, and purified by octyl-Sepharose 4B (4). FerredoxinNAp activity was eluted at a potassium chloride concentration of about 0.2 M. Fractions containing ferredoxinNAP were pooled, dialyzed overnight, and concentrated over an Amicon filter (YM5 membrane). The concentrated solution was applied to a Sephadex G-75 superfine column (2.6 by 90 cm), and proteins were eluted with TEG buffer. Fractions from the Sephadex column containing ferredoxinNAp activity were pooled and applied to a DEAE-cellulose column (1 by 8 cm). The column was washed with 50 ml of buffer, and the protein was eluted with a 100-ml continuous KCl gradient (0 to 800 mM) in TEG buffer. A single protein peak which coincided
with ferredoxinNAp activity eluted at about 0.3 M KCl. Fractions containing ferredoxinNAp activity were pooled, dialyzed against TEG buffer for 3 h, and stored at -20°C. Although the protein yield was low (3.2 mg), these procedures resulted in greater than 300-fold purification of the ferredoxinNAp after Blue Sepharose CL-6B chromatography (Table 1). Only 21% of the activity originally present in the Blue Sepharose eluate was recovered. The specific activity was 3,375 nmol of naphthalene oxidized per min per mg of ferredoxinNAP. Sodium dodecyl sulfate (SDS)-polyacrylamide gradient (10 to 20%) gel (Isolab, Inc., Akron, Ohio) electrophoresis revealed a single band that stained for protein. The molecular weight of ferredoxinNAp determined by SDS-polyacrylamide gradient gel electrophoresis and native gel electrophoresis was 13,000 and 14,400, respectively. Determination of the molecular weight from the amino acid composition gave
20
c
E 16 0
am 0
Ea
a
12
IL N
a
TABLE 3. Substitution of ferredoxinNAp reductase by spinach ferredoxin reductase in naphthalene dioxygenase assays' Sp act (,umol/mg) Reductase
Spinach ferredoxin reductase
FerredoxinNAp reductase
Addition
NADH NADPH NADH NADPH
Without FAD
With
0.250 0.338 13.2 1.52
0.252 0.50 71.0 36.7
FAD
a Naphthalene dioxygenase assays were performed with saturating levels of
ferredoxinNAp and ISPNAP and either NADH or NADPH (1.0 ,umol), with and without FAD (1.0 nmol). Specific activity is micromoles of naphthalene oxidized per minute per milligram of reductase component.
0
w
8
z -j
4
I
z 0.
0
1
2
FEREDOXINNAp
3
4
5
(ug of protein)
FIG. 2. Requirement of ferredoxinNAP for naphthalene dioxygenase activity. Naphthalene dioxygenase activity was determined as described previously (5), with partially purified ISPNAP (150 ,ug), purified ferredoxinNAp reductase (20 ,ug), and the indicated amounts of purified ferredoxinNAP-
VOL. 172, 1990
NOTES
467
OH
NDH + H+
Rductase ( (ox)
N+
ReductaseNW (red)
X
Ferredoxin NAP (red)
Ferredoxin NAP (ox)
x
(O (ox)X
ISPX (red)
FIG. 3. Proposed electron transport scheme for naphthalene dioxygenase. ox, Oxidized; red, reduced.
a value of 13,402 (Table 2). The average of these three methods (Mr 13,600) was used in subsequent calculations. The isoelectric point of ferredoxinNAp determined in ampholyte vertical slab gels (pH 3 to 5) was pH 4.6. Solutions of ferredoxinNAP were brown, and the oxidized spectrum gave absorption maxima at 280, 325, and 460 nm, with a broad shoulder at 550 nm (Fig. 1). Extinction coefficients calculated at 325 and 460 nm were 9.85 mM` cm` and 4.92 mM-1 cm-', respectively. Iron and acid-labile sulfur analysis showed that ferredoxinNAP contained 1.7 g-atoms of iron and 2.1 g-atoms of acid-labile sulfur per mol of enzyme. FerredoxinNAp reductase reduces ferredoxinNAP in the presence of NADH and FAD (9). Spinach ferredoxin reductase (Sigma Chemical Co., Rockford, Ill.) catalyzed cytochrome c reductase activity in the presence of ferredoxinNAp and NADH or NADPH but not in controls in which either of the protein components were omitted. Spinach ferredoxin reductase substituted for reductaseNAP in naphthalene dioxygenase assays, but it was significantly less active (Table 3). The activities with NADPH were higher than those with NADH as an electron donor in assays containing spinach ferredoxin reductase. In assays containing ferredoxinNAp reductase, the activity was highest when NADH served as the electron donor. Naphthalene dioxygenase activity was not observed in the absence of one or more of the protein components. In a similar study, replacement of ferredoxinTOL reductase with spinach ferredoxin reductase in the toluene dioxygenase system resulted in significant activity with NADPH as the electron donor but not NADH (7). Naphthalene dioxygenase activity was observed only in the presence of ferredoxinNAP reductase, ferredoxinNAP, and 'SPNAP (Fig. 2). In the absence of ferredoxinNAP, no naphthalene dioxygenase activity was observed. Analogous electron transfer proteins such as spinach ferredoxin (type III) (13), ferredoxin from Clostridium pasteurianum (type V) (11), ferredoxin from a red marine alga (Porphyra umbilicus) (type VI) (1), ferredoxinTOL (16), and putidaredoxin (8) could not replace ferredoxinNAP in the dioxygenase system. FerredoxinNAP is a red-brown protein with spectral properties similar to those of the ferredoxin proteins purified from benzene dioxygenase (2, 3), toluene dioxygenase (16), and the Rieske iron-sulfur protein from Thermus thermophilus (6). The calculated extinction value of ferredoxinNAP at 460 nm (146oS 4.92 mM1 cm-) was approximately one-half of that reported for 'SPNAP (E462, 9.0 mM1 Cm1) at 462 nm (4). ISPNAP exhibits a spectrum similar to that of ferredoxinNAP, with absorption maxima in the visible region at 566 (shoulder), 462, and 334 nm (4). This protein has been suggested to contain two [2Fe-2S] clusters with additional iron atoms required for activity (4). The similarities in the spectral characteristics of these proteins suggest that the iron-sulfur chromophore present in ferredoxinNAP is arranged in an environment similar to those of 'SPNAP.
The size of ferredoxinNAP is comparable to that reported for ferredoxinTOL, Mr 15,300 (16); the ferredoxin from the benzene dioxygenase system, Mr 12,300 (2); putidaredoxin, Mr 12,500 (17); the ferredoxin from pyrazon dioxygenase, Mr 12,000 (14); and the plant-type ferredoxins (12). The properties of ferredoxinNAp are similar to those of other plant and bacterial ferredoxins which are small acidic proteins, containing a [2Fe-2S] cluster (12). The results from this and previous studies indicate that ferredoxinNAP functions as an intermediate electron transfer protein and support the proposed arrangement of the components in the naphthalene dioxygenase system shown in Fig. 3 (4, 5, 9). Although spinach ferredoxin reductase partially substituted for ferredoxinNAp reductase in the naphthalene dioxygenase assay, spinach ferredoxin (13) did not replace ferredoxinNAP in this system. Similar results were obtained with other analogous electron transfer proteins. These observations indicate that there is a specific interaction between ferredoxinNAp and ISPNAP during electron transfer. From similar substitution studies, such interactions with their respective oxygenases have been suggested for ferredoxinTOL (16), putidaredoxin (8), and adrendoxin (10). With purified enzyme components available, the study of this complex protein and the function of each redox center can now be undertaken. We thank Venkiteswaran Subramanian and Irwin C. Gunsalus for providing us with ferredoxinTOL and putidaredoxin, respectively. We also thank Burt Ensley and Wen Chen Suen for helpful discussions, Shirley F. Nishino for assistance in preparation of the manuscript, and Jim C. Spain for reviewing the manuscript. This work was supported by Public Health Service grant GM29909 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Andrew, P. W., L. J. Roger&, D. Boulter, and B. G. Haslett. 1976. Ferredoxin from a red alga, Porphyra umbilicalis. Eur. J. Biochem. 69:243-248. 2. Axcell, B. C., and P. J. Geary. 1975. Purification and some properties of a soluble benzene-oxidizing system from a strain of Pseudomonas. Biochem. J. 146:173-183. 3. Crutcher, S. E., and P. J. Geary. 1979. Properties of the iron-sulfur proteins of the benzene dioxygenase system from Pseudomonas putida. Biochem. J. 177:393-400. 4. Ensley, B. D., and D. T. Gibson. 1983. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. J. Bacteriol. 155:505-511. 5. Ensley, B. D., D. T. Gibson, and A. L. Laborde. 1982. Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 149:948-954. 6. Fee, J. A., K. L. Findling, T. Yoshida, R. Hille, G. E. Tarr, D. 0. Hearshen, W. R. Dunham, E. P. Day, T. A. Kent, and E. Munck. 1984. Purification and characterization of the Rieske iron-sulfur protein from Thermus thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine ligands. J. Biol. Chem. 259:124-133. 7. Gibson, D. T., W.-K. Yeh, T.-N. Liu, and V. Subramanian. 1982. Toluene dioxygenase: a multicomponent enzyme system from Pseudomonas putida, p. 51-61. In M. Nozaki, S. Yama-
468
8.
9.
10. 11.
12.
NOTES moto, Y. Ishimura, M. J. Coon, L. Ernster, and R. W. Estabrook (ed.), Oxygenases and oxygen metabolism. Academic Press, Inc., New York. Gunsalus, I. C., and J. D. Lipscomb. 1973. Structure and reactions of a microbial monooxygenase: the role of putidaredoxin, p. 151-171. In W. Lovenberg (ed.), Iron-sulfur proteins, vol. 1. Academic Press, Inc., New York. Haigler, B. E., and D. T. Gibson. 1990. Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 172:457-464. Huang, J. J., and T. Kimura. 1971. A specific role of reduced adrenodoxin in adrenal mitochondrial steroid hydroxylases. Biochem. Biophys. Res. Commun. 44:1065-1070. Lovenberg, W., B. B. Buchanan, and J. C. Rabinowitz. 1963. Studies on the chemical nature of clostridial ferredoxin. J. Biol. Chem. 238:3899-3913. Malkin, R. 1973. The chemical properties of ferredoxins, p. 1-26. In W. Lovenberg (ed.), Iron-sulfur proteins, vol 2. Aca-
J. BACTERIOL.
demic Press, Inc., New York. 13. Matsubara, H., R. M. Sasaki, and R. K. Chain. 1968. Spinach ferredoxin: amino acid composition and terminal sequences. J. Biol. Chem. 243:1725-1731. 14. Sauber, K., C. Frohner, G. Rosenberg, J. Eberspacher, and F. Lingens. 1977. Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria. Eur. J. Biochem. 74: 89-97. 15. Spencer, R. L., and F. Wold. 1969. A new convenient method for estimation of total cystine-cysteine in proteins. Anal. Biochem. 32:185-190. 16. Subramanian, V., T.-N. Liu, W.-K. Yeh, C. M. Serdar, L. P. Wackett, and D. T. Gibson. 1985. Purification and properties of ferredoxinTOL, a component of toluene dioxygenase from Pseudomonas putida Fl. J. Biol. Chem. 260:2355-2363. 17. Tsai, R. L., I. C. Gunsalus, and K. Dus. 1971. Composition and structure of camphor hydroxylase components and homology between putidaredoxin and adrenodoxin. Biochem. Biophys. Res. Commun. 45:1300-1306.
Vol. 172, No. 1
NOTES Purification and Properties of FerredoxinNAP, a Component of Naphthalene Dioxygenase from Pseudomonas sp. Strain NCIB 9816 BILLY E. HAIGLERt* AND DAVID T. GIBSONt Center for Applied Microb ology and Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received 19 May 1989/Accepted 2 October 1989
One of the three components of the naphthalene dioxygenase occurring in induced cells of Pseudomonas sp. strain NCIB 9816 has been purified to homogeneity. The protein contained 2 g-atoms each of iron and acid-labile sulfur and had an apparent molecular weight of 13,600. The evidence indicates that it is a ferredoxin-type protein that functions as an intermediate electron transfer protein in naphthalene dioxygenase activity.
Naphthalene dioxygenase is a multicomponent enzyme system which catalyzes the oxidation of naphthalene to cis-(lR,2S)-dihydroxy-1,2-dihydronaphthalene (4, 5). Naphthalene dioxygenase consists of three enzymes previously designated A, B, and C (5). Purification and characterization of component B showed that it was an iron-sulfur protein, designated 'SPNAP, that functioned as the terminal oxygenase (4). ISPNAP catalyzes the insertion of dioxygen into the aromatic ring of naphthalene (4). The ability of component A to transfer electrons from NADH to cytochrome c indicated that it was an NADH-oxidoreductase (4, 5). The activity of component A in the naphthalene dioxygenase and cytochrome c reductase assays was enhanced in the presence of flavin adenine dinucleotide (FAD) or flavin mononucleotide, with FAD being the preferred flavin cofactor (5, 9). Component A was shown to be an iron-sulfur flavoprotein which acts as the initial electron acceptor to shuttle electons from NADH to the terminal oxygenase (4, 5, 9). The function of component C was not clear, but it appeared to be an intermediate electron carrier in the enzyme system (4, 5). It was necessary to purify each of these proteins to establish their roles in naphthalene dioxygenation. Here we report the purification and characterization of ferredoxinNAP, previously designated component C. The results indicate that ferredoxinNAP is an iron-sulfur protein similar to the ferredoxin that functions in the toluene dioxygenase system (ferredoxinTOL) (16) and the ferredoxin component of the benzene dioxygenase system (2, 3). In another report, we show that ferredoxinNAP reductase, previously designated component A, can transfer electrons from NADH to ferredoxinNAP (9), thus supporting the role of ferredoxinNAp as an intermediate electron carrier in the naphthalene dioxygenase system. Pseudomonas sp. strain NCIB 9816 was grown and maintained as described previously (9). Lysis of the cells, protein and analytical determinations, and separation of ferre*
TABLE 1. Purification of ferredoxinNApa Purification step
Total Activity Sp act Purification protein (U) (U/mg) (fold) (mg)
Blue Sepharose 4,880 52,400 10.7 DEAE-cellulose 595 35.7 21,280 5.6 13,246 2,365 Sephadex G-75 DEAE-cellulose 3.2 10,800 3,375 a Details of the
purification procedure
Recovery (%)
100 41 25 21
3 221 315
are described in the text.
FerredoxinNAp-dependent naphthalene dioxygenase activity was measured in the presence of saturating levels of ferredoxinNAp reductase and ISPNAP. One unit of activity is defined as the amount of ferredoxinNAp required to oxidize 1.0 nmol of naphthalene per min.
doxinNAp and ISPNAP from NADH-ferredoxinNAP reductase by Blue Sepharose CL-6B gel chromatography were done as described before (9). Except where noted, all media, chemicals, and enzymes were those used in previous investigations (9, 16). Iron, amino acid, and acid-labile sulfur determinations were performed as previously described (9). FerredoxinNAP was located in column eluates by its ability to TABLE 2. Amino acid composition of ferredoxinNAP Amino acid
Lysine Arginine Threoninea Glycine Half-cystine" Methioninec Leucine
Phenylalanine Aspartate + asparagine
Corresponding author.
No. of residues/mol of enzyme
8 4 8 9 4 3 14 3 12
Amino acid
Histidine Serinea Proline Alanine Valine Isoleucine
Tyrosine Tryptophan Glutamate +
No. of residues/mol of enzyme
2 5 4
10 11 7 3 NDd 15
glutamine
a Determined by extrapolation to zero time of hydrolysis. b Determined after 24 h of hydrolysis in the presence of dimethyl sulfoxide
t Present address: Air Force Engineering and Services Center, Tyndall Air Force Base, FL 32403. t Present address: Department of Microbiology, College of Medicine, University of Iowa, Iowa City, IA 52242.
(15). ' d
465
Determined as methionine sulfone. ND, Not determined.
466
J. BACTERIOL.
NOTES
2.0
1.5
0.1
0.5
0.0
550
450
350
250
650
WAVELENGTH (nm) FIG. 1. Absorption spectrum of oxidized ferredoxinNAP. The vertical line indicates where the absorbance scale was changed.
catalyze naphthalene dioxygenase activity in the presence of
ferredoxinNAp reductase, ISPNAP, FAD, and NADH. Protein purification was carried out at 4°C. Fractions containing 'SPNAP and ferredoxinNAp activity eluted from the Blue Sepharose CL-6B column in the void volume and a portion of the buffer wash (9). The fractions containing ferredoxinNAp activity from two such column purifications (representing 100 g of cells [wet weight]) were pooled, centrifuged to remove particulate matter, and loaded on a DEAE-cellulose column (4 by 12 cm). The column was washed with 1 liter of TEG buffer (50 mM Tris hydrochloride buffer [pH 7.5] containing 10% [vol/vol] ethanol, 10% [vol/ vol] glycerol, and 0.5 mM dithiothreitol), and ISPNAP and ferredoxinNAP were separated with a continuous KCI gradient (0.0 to 0.4 M KCl) in 1 liter of TEG buffer. Fractions containing ISPNAP activity were pooled, concentrated, and purified by octyl-Sepharose 4B (4). FerredoxinNAp activity was eluted at a potassium chloride concentration of about 0.2 M. Fractions containing ferredoxinNAP were pooled, dialyzed overnight, and concentrated over an Amicon filter (YM5 membrane). The concentrated solution was applied to a Sephadex G-75 superfine column (2.6 by 90 cm), and proteins were eluted with TEG buffer. Fractions from the Sephadex column containing ferredoxinNAp activity were pooled and applied to a DEAE-cellulose column (1 by 8 cm). The column was washed with 50 ml of buffer, and the protein was eluted with a 100-ml continuous KCl gradient (0 to 800 mM) in TEG buffer. A single protein peak which coincided
with ferredoxinNAp activity eluted at about 0.3 M KCl. Fractions containing ferredoxinNAp activity were pooled, dialyzed against TEG buffer for 3 h, and stored at -20°C. Although the protein yield was low (3.2 mg), these procedures resulted in greater than 300-fold purification of the ferredoxinNAp after Blue Sepharose CL-6B chromatography (Table 1). Only 21% of the activity originally present in the Blue Sepharose eluate was recovered. The specific activity was 3,375 nmol of naphthalene oxidized per min per mg of ferredoxinNAP. Sodium dodecyl sulfate (SDS)-polyacrylamide gradient (10 to 20%) gel (Isolab, Inc., Akron, Ohio) electrophoresis revealed a single band that stained for protein. The molecular weight of ferredoxinNAp determined by SDS-polyacrylamide gradient gel electrophoresis and native gel electrophoresis was 13,000 and 14,400, respectively. Determination of the molecular weight from the amino acid composition gave
20
c
E 16 0
am 0
Ea
a
12
IL N
a
TABLE 3. Substitution of ferredoxinNAp reductase by spinach ferredoxin reductase in naphthalene dioxygenase assays' Sp act (,umol/mg) Reductase
Spinach ferredoxin reductase
FerredoxinNAp reductase
Addition
NADH NADPH NADH NADPH
Without FAD
With
0.250 0.338 13.2 1.52
0.252 0.50 71.0 36.7
FAD
a Naphthalene dioxygenase assays were performed with saturating levels of
ferredoxinNAp and ISPNAP and either NADH or NADPH (1.0 ,umol), with and without FAD (1.0 nmol). Specific activity is micromoles of naphthalene oxidized per minute per milligram of reductase component.
0
w
8
z -j
4
I
z 0.
0
1
2
FEREDOXINNAp
3
4
5
(ug of protein)
FIG. 2. Requirement of ferredoxinNAP for naphthalene dioxygenase activity. Naphthalene dioxygenase activity was determined as described previously (5), with partially purified ISPNAP (150 ,ug), purified ferredoxinNAp reductase (20 ,ug), and the indicated amounts of purified ferredoxinNAP-
VOL. 172, 1990
NOTES
467
OH
NDH + H+
Rductase ( (ox)
N+
ReductaseNW (red)
X
Ferredoxin NAP (red)
Ferredoxin NAP (ox)
x
(O (ox)X
ISPX (red)
FIG. 3. Proposed electron transport scheme for naphthalene dioxygenase. ox, Oxidized; red, reduced.
a value of 13,402 (Table 2). The average of these three methods (Mr 13,600) was used in subsequent calculations. The isoelectric point of ferredoxinNAp determined in ampholyte vertical slab gels (pH 3 to 5) was pH 4.6. Solutions of ferredoxinNAP were brown, and the oxidized spectrum gave absorption maxima at 280, 325, and 460 nm, with a broad shoulder at 550 nm (Fig. 1). Extinction coefficients calculated at 325 and 460 nm were 9.85 mM` cm` and 4.92 mM-1 cm-', respectively. Iron and acid-labile sulfur analysis showed that ferredoxinNAP contained 1.7 g-atoms of iron and 2.1 g-atoms of acid-labile sulfur per mol of enzyme. FerredoxinNAp reductase reduces ferredoxinNAP in the presence of NADH and FAD (9). Spinach ferredoxin reductase (Sigma Chemical Co., Rockford, Ill.) catalyzed cytochrome c reductase activity in the presence of ferredoxinNAp and NADH or NADPH but not in controls in which either of the protein components were omitted. Spinach ferredoxin reductase substituted for reductaseNAP in naphthalene dioxygenase assays, but it was significantly less active (Table 3). The activities with NADPH were higher than those with NADH as an electron donor in assays containing spinach ferredoxin reductase. In assays containing ferredoxinNAp reductase, the activity was highest when NADH served as the electron donor. Naphthalene dioxygenase activity was not observed in the absence of one or more of the protein components. In a similar study, replacement of ferredoxinTOL reductase with spinach ferredoxin reductase in the toluene dioxygenase system resulted in significant activity with NADPH as the electron donor but not NADH (7). Naphthalene dioxygenase activity was observed only in the presence of ferredoxinNAP reductase, ferredoxinNAP, and 'SPNAP (Fig. 2). In the absence of ferredoxinNAP, no naphthalene dioxygenase activity was observed. Analogous electron transfer proteins such as spinach ferredoxin (type III) (13), ferredoxin from Clostridium pasteurianum (type V) (11), ferredoxin from a red marine alga (Porphyra umbilicus) (type VI) (1), ferredoxinTOL (16), and putidaredoxin (8) could not replace ferredoxinNAP in the dioxygenase system. FerredoxinNAP is a red-brown protein with spectral properties similar to those of the ferredoxin proteins purified from benzene dioxygenase (2, 3), toluene dioxygenase (16), and the Rieske iron-sulfur protein from Thermus thermophilus (6). The calculated extinction value of ferredoxinNAP at 460 nm (146oS 4.92 mM1 cm-) was approximately one-half of that reported for 'SPNAP (E462, 9.0 mM1 Cm1) at 462 nm (4). ISPNAP exhibits a spectrum similar to that of ferredoxinNAP, with absorption maxima in the visible region at 566 (shoulder), 462, and 334 nm (4). This protein has been suggested to contain two [2Fe-2S] clusters with additional iron atoms required for activity (4). The similarities in the spectral characteristics of these proteins suggest that the iron-sulfur chromophore present in ferredoxinNAP is arranged in an environment similar to those of 'SPNAP.
The size of ferredoxinNAP is comparable to that reported for ferredoxinTOL, Mr 15,300 (16); the ferredoxin from the benzene dioxygenase system, Mr 12,300 (2); putidaredoxin, Mr 12,500 (17); the ferredoxin from pyrazon dioxygenase, Mr 12,000 (14); and the plant-type ferredoxins (12). The properties of ferredoxinNAp are similar to those of other plant and bacterial ferredoxins which are small acidic proteins, containing a [2Fe-2S] cluster (12). The results from this and previous studies indicate that ferredoxinNAP functions as an intermediate electron transfer protein and support the proposed arrangement of the components in the naphthalene dioxygenase system shown in Fig. 3 (4, 5, 9). Although spinach ferredoxin reductase partially substituted for ferredoxinNAp reductase in the naphthalene dioxygenase assay, spinach ferredoxin (13) did not replace ferredoxinNAP in this system. Similar results were obtained with other analogous electron transfer proteins. These observations indicate that there is a specific interaction between ferredoxinNAp and ISPNAP during electron transfer. From similar substitution studies, such interactions with their respective oxygenases have been suggested for ferredoxinTOL (16), putidaredoxin (8), and adrendoxin (10). With purified enzyme components available, the study of this complex protein and the function of each redox center can now be undertaken. We thank Venkiteswaran Subramanian and Irwin C. Gunsalus for providing us with ferredoxinTOL and putidaredoxin, respectively. We also thank Burt Ensley and Wen Chen Suen for helpful discussions, Shirley F. Nishino for assistance in preparation of the manuscript, and Jim C. Spain for reviewing the manuscript. This work was supported by Public Health Service grant GM29909 from the National Institute of General Medical Sciences.
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