Proc. Natl. Acad. Sci. USA Vol. 87, pp. 8%5-8969, November 1990 Biochemistry
Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: Modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster (electron paramagnetic resonance/iron-sulfur protein/valence delocalization/succinate dehydrogenase/hydrogen bonding)
MARK T. WERTH*, GARY CECCHINIt, ANAMARIA MANODORIt, BRIAN A. C. ACKRELLt, IMKE ROBERT P. GUNSALUS*, AND MICHAEL K. JOHNSON*§
SCHRODERt,
*Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602; tMolecular Biology Division, Veterans Administration Medical Center, San Francisco, CA 94121, and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143; and tDepartment of Microbiology and Molecular Genetics and Molecular Biology Institute, University of California, Los Angeles, CA 90024
Communicated by Helmut Beinert, August 27, 1990 (received for review June 13, 1990)
ABSTRACT Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers. In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., center 1, [2Fe-2S]; center 2, [4Fe-4S]; center 3, [3Fe-4S]) have been assembled. The mutations affect the properties of center 1 only and demonstrate that the anomalously high redox potential of this [2Fe-2S] center is essential for optimal enzymatic activity. The results are consistent with cysteines 57, 62, 65, and 77 providing the ligands to center 1 but leave open the possibility of noncysteinyl coordination for the localized valence Fe(Hi) site of the reduced cluster. The implications of the results for the role of center 1 in the electron-transfer pathway and the valence localization of reduced center 1 are discussed.
The menaquinol-fumarate oxidoreductase (EC 1.3.99.1) of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor (1, 2). The membrane-extrinsic fumarate reductase domain comprises a flavoprotein (Fp), FrdA (66 kDa), with a covalently bound FAD (3), and an iron-sulfur protein (Ip), FrdB (27 kDa). Two small hydrophobic peptides (4), FrdC (15 kDa) and FrdD (13 kDa), anchor the enzyme to the membrane and are essential for interaction with quinones (5-7). The combination of magnetic CD and EPR spectroscopies has provided evidence for three types of iron-sulfur clusters, each stoichiometric with FAD, in the two-subunit fumarate reductase: center 1, [2Fe2SI2+ 1+; center 2, [4Fe-4SJ2+"1; center 3, [3Fe4S]J+ ° (811). Centers 1 and 3 have been localized within the FrdB subunit, whereas center 2 requires the presence of the FrdA peptide for assembly, and thus may be coordinated by amino acid residue(s) from the FrdA subunit (12). The enzyme complex is also catalytically competent for succinate oxidation and, accordingly, is very similar in terms of number, type, and properties of the iron-sulfur centers and subunit composition to tetrameric succinate dehydrogenase complexes in both prokaryotic and eukaryotic cells (13, 14). Comparison of the six amino acid sequences of the Ip subunits of fumarate reductases and succinate dehydrogenases that have been determined thus far reveals a high degree of homology, with 11 conserved cysteine residues (10 in E. coli succinate dehydrogenase) in three ferredoxin-like clus-
E. coli P. vulgaris W. succinogenes Bovine Heart B. sublilis E. coli S. platensis
Frdlp Frdlp Frdlp SdhIp Sdhlp SdhIp 2Fe Fd
56-S 57-S 55-V 64-S 62-N 53-S 39-S
RMAI CGS GMMVNNVPKLAc RMAI CGS CGMMVNRVPKLA C RAGI GS C GMMINGRPSLAJC REGI S CAMNINGGNTLAC LEEV
REGV RAGA
GASMVINGKPRQSC GS GLNMNGKNGLAU T CA
(26Residues)
LTC
FIG. 1. Comparison of the arrangement of N-terminal cysteine residues in the Ip subunits of fumarate reductases (Frd) from E. coli (16), Proteus vulgaris (19), and Wolinella succinogenes (20) and succinate dehydrogenases (Sdh) from bovine heart (15), Bacillus subtilis (18), and E. coli (17), with those of the 2Fe ferredoxin (Fd) from Spirulina platensis (21).
ters (13-20). Furthermore, sequence comparisons with the structurally characterized chloroplast-type [2Fe-2S] ferredoxin from Spirulina platensis (21) (Fig. 1), and EPR studies of mutant enzymes with truncated Ip subunits (12, 22), suggest that center 1 is ligated by the group of four conserved cysteine residues closest to the N terminus. However, there are two puzzling aspects of this assignment. First, the third cysteine residue in this grouping is replaced by an aspartic residue in E. coli succinate dehydrogenase (17) (Fig. 1). Second, although the EPR signals of reduced center 1 are typical of ferredoxin-type [2Fe-2S]'+ clusters-i.e., gav = 1.96 (23)-their midpoint potentials are between 200 and 500 mV more positive (13, 14), and in the range observed for Rieske-type [2Fe-2S] centers in Thermus thermophilus HB-8 Rieske protein andibacterial dioxygenases, which have histidinyl coordination at the localized valence Fe(II) site (24). To address these problems and provide more definitive evidence for the amino acid ligands to center 1, we have characterized the EPR and redox properties of site-directed mutants of E. coli fumarate reductase in which each of the first'four conserved cysteine residues in the FrdB subunit is selectively replaced by a serine residue.
MATERIALS AND METHODS Strains, Plasmids, and Phage. The E. coli strains, plasmids, and phage used in this work are listed in Table 1. Strain DW12 contains a deletion of the frdABCD region in the E. coli chromosome (29). Phage M13KWI was constructed by inserting the 2.0-kilobase (kb) EcoRI-Sal I fragment from pH3 into the polylinker site of M13mp8. Site-Directed Mutagenesis. Site-directed mutagenesis was performed using the in vitro mutagenesis system from Amersham based on the method developed by Eckstein and Abbreviations: Fp, flavoprotein; Ip, iron-sulfur protein; PMS, phenazine methosulfate; BVrd, reduced benzyl viologen; MQH2, menaquinol-6. §To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8965
8966
Biochemistry: Werth et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
Table 1. E. coli strains, plasmids, and phage Genotype Origin Strain Bacteria F- zjd::TnlO A(frdABCD)102 araDI39 A(argF-4ac) U169 rpsL50 MC4100 DW12 relAI flbBS301 deoCI pfsF2S rbsR F- 480d/lacZAMJ5 endAI recAl hsdRJ7(rk-,mk+) supE44 thi-J DH5a gyrA relAI A(lacZYA-argF) U169 ATG1 A(lac-pro) supE thi hsdD5/F' tra36 proA+B+ IacP I-acZAMJ5 Plasmids pBR322 frdA+lB+C+D+ pH3 pH3 pH3FrdBC57S frdA+Bc57sC+D+ pH3 pH3FrdBC62S frdA+Bc62sC+D+ frdA+Bc6ssC+D+ pH3 pH3FrdBC65S pH3 pH3FrdBC77S frdA+Bc77sC+D+ Phage M13mp8 frdA'B+C' M13mp8 M13KW1
coworkers (30). Oligonucleotides for mutagenesis and sequencing were synthesized on a Biosearch model 8700. The oligonucleotides differ from the wild type by having one nucleotide altered, thus changing cysteine residues 57, 62, 65, and 77 to serine residues. The mutagenesis was performed using single-stranded M13KWI DNA as a template. The mutants were identified by DNA sequence analysis using the dideoxy termination proce ure of Sanger et al. (25) (Pharmacia sequencing kit). Strain TG1 was used for mutagenesis and single-stranded DNA sequence analysis. Following mutagenesis, the 2.0-kb EcoRI-Sal I fragment containing the frdB region was cloned back into the large EcoRI-Sal I fragment of plasmid pH3 to restore the complete frdABCl) operon. The mutation in this construct was confirmed by double-stranded DNA sequencing, using strain DH5a to prepare DNA. DNA was prepared using the Qiagen plasmid kit from Diagen (Chatsworth, CA). The four frdB mutant plasmids are designated pH3FrdBC57S, pH3FrdBC62S, pH3FrdBC65S, and pH3FrdBC77S (Table 1). Growth of Bacteria. For biochemical analysis the mutant plasmids were transformed into DW12 [A(frdABCD)]. The E. coli strains were grown anaerobically on glycerol/fumarate, glycerol/nitrate, and glucose/fumarate minimal media as described (6). Cells used for preparation of the membrane fraction and purified enzyme complexes were grown anaerobically on glucose/fumarate medium to stationary phase, chilled, and harvested by centrifugation. For phage and plasmid manipulations, cells were grown on Luria broth (31) and' solid media. Purification of Membranes Enriched in Fumarate Reductase. Sixty grams (wet weight) of E. coli DW12 containing the appropriate plasmid was suspended in 300 ml of 30 mM Tris/HCl (pH 8.0). The inner-membrane/fumarate reductase tubule fraction was then purified as described (32). The membranes obtained were enriched 2- to 3-fold for fumarate reductase, based on assay of covalent flavin {8a-[N(3)-histidyl] FAD where N(3) is Ni1, over the crude membrane preparations previously used (6). Enzyme Assays. Fumarate-dependent oxidation of reduced benzyl viologen (BVred) and menaquinol-6 (MQH2) and succinate-dependent reduction of phenazine methosulfate (PMS) were determined as described (6). EPR Spectroscopy. Samples for EPR spectroscopy were prepared under an argon atmosphere in a Vacuum Atmospheres (Hawthorne, CA) glove box (
Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: Modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster (electron paramagnetic resonance/iron-sulfur protein/valence delocalization/succinate dehydrogenase/hydrogen bonding)
MARK T. WERTH*, GARY CECCHINIt, ANAMARIA MANODORIt, BRIAN A. C. ACKRELLt, IMKE ROBERT P. GUNSALUS*, AND MICHAEL K. JOHNSON*§
SCHRODERt,
*Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602; tMolecular Biology Division, Veterans Administration Medical Center, San Francisco, CA 94121, and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143; and tDepartment of Microbiology and Molecular Genetics and Molecular Biology Institute, University of California, Los Angeles, CA 90024
Communicated by Helmut Beinert, August 27, 1990 (received for review June 13, 1990)
ABSTRACT Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers. In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., center 1, [2Fe-2S]; center 2, [4Fe-4S]; center 3, [3Fe-4S]) have been assembled. The mutations affect the properties of center 1 only and demonstrate that the anomalously high redox potential of this [2Fe-2S] center is essential for optimal enzymatic activity. The results are consistent with cysteines 57, 62, 65, and 77 providing the ligands to center 1 but leave open the possibility of noncysteinyl coordination for the localized valence Fe(Hi) site of the reduced cluster. The implications of the results for the role of center 1 in the electron-transfer pathway and the valence localization of reduced center 1 are discussed.
The menaquinol-fumarate oxidoreductase (EC 1.3.99.1) of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor (1, 2). The membrane-extrinsic fumarate reductase domain comprises a flavoprotein (Fp), FrdA (66 kDa), with a covalently bound FAD (3), and an iron-sulfur protein (Ip), FrdB (27 kDa). Two small hydrophobic peptides (4), FrdC (15 kDa) and FrdD (13 kDa), anchor the enzyme to the membrane and are essential for interaction with quinones (5-7). The combination of magnetic CD and EPR spectroscopies has provided evidence for three types of iron-sulfur clusters, each stoichiometric with FAD, in the two-subunit fumarate reductase: center 1, [2Fe2SI2+ 1+; center 2, [4Fe-4SJ2+"1; center 3, [3Fe4S]J+ ° (811). Centers 1 and 3 have been localized within the FrdB subunit, whereas center 2 requires the presence of the FrdA peptide for assembly, and thus may be coordinated by amino acid residue(s) from the FrdA subunit (12). The enzyme complex is also catalytically competent for succinate oxidation and, accordingly, is very similar in terms of number, type, and properties of the iron-sulfur centers and subunit composition to tetrameric succinate dehydrogenase complexes in both prokaryotic and eukaryotic cells (13, 14). Comparison of the six amino acid sequences of the Ip subunits of fumarate reductases and succinate dehydrogenases that have been determined thus far reveals a high degree of homology, with 11 conserved cysteine residues (10 in E. coli succinate dehydrogenase) in three ferredoxin-like clus-
E. coli P. vulgaris W. succinogenes Bovine Heart B. sublilis E. coli S. platensis
Frdlp Frdlp Frdlp SdhIp Sdhlp SdhIp 2Fe Fd
56-S 57-S 55-V 64-S 62-N 53-S 39-S
RMAI CGS GMMVNNVPKLAc RMAI CGS CGMMVNRVPKLA C RAGI GS C GMMINGRPSLAJC REGI S CAMNINGGNTLAC LEEV
REGV RAGA
GASMVINGKPRQSC GS GLNMNGKNGLAU T CA
(26Residues)
LTC
FIG. 1. Comparison of the arrangement of N-terminal cysteine residues in the Ip subunits of fumarate reductases (Frd) from E. coli (16), Proteus vulgaris (19), and Wolinella succinogenes (20) and succinate dehydrogenases (Sdh) from bovine heart (15), Bacillus subtilis (18), and E. coli (17), with those of the 2Fe ferredoxin (Fd) from Spirulina platensis (21).
ters (13-20). Furthermore, sequence comparisons with the structurally characterized chloroplast-type [2Fe-2S] ferredoxin from Spirulina platensis (21) (Fig. 1), and EPR studies of mutant enzymes with truncated Ip subunits (12, 22), suggest that center 1 is ligated by the group of four conserved cysteine residues closest to the N terminus. However, there are two puzzling aspects of this assignment. First, the third cysteine residue in this grouping is replaced by an aspartic residue in E. coli succinate dehydrogenase (17) (Fig. 1). Second, although the EPR signals of reduced center 1 are typical of ferredoxin-type [2Fe-2S]'+ clusters-i.e., gav = 1.96 (23)-their midpoint potentials are between 200 and 500 mV more positive (13, 14), and in the range observed for Rieske-type [2Fe-2S] centers in Thermus thermophilus HB-8 Rieske protein andibacterial dioxygenases, which have histidinyl coordination at the localized valence Fe(II) site (24). To address these problems and provide more definitive evidence for the amino acid ligands to center 1, we have characterized the EPR and redox properties of site-directed mutants of E. coli fumarate reductase in which each of the first'four conserved cysteine residues in the FrdB subunit is selectively replaced by a serine residue.
MATERIALS AND METHODS Strains, Plasmids, and Phage. The E. coli strains, plasmids, and phage used in this work are listed in Table 1. Strain DW12 contains a deletion of the frdABCD region in the E. coli chromosome (29). Phage M13KWI was constructed by inserting the 2.0-kilobase (kb) EcoRI-Sal I fragment from pH3 into the polylinker site of M13mp8. Site-Directed Mutagenesis. Site-directed mutagenesis was performed using the in vitro mutagenesis system from Amersham based on the method developed by Eckstein and Abbreviations: Fp, flavoprotein; Ip, iron-sulfur protein; PMS, phenazine methosulfate; BVrd, reduced benzyl viologen; MQH2, menaquinol-6. §To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8965
8966
Biochemistry: Werth et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
Table 1. E. coli strains, plasmids, and phage Genotype Origin Strain Bacteria F- zjd::TnlO A(frdABCD)102 araDI39 A(argF-4ac) U169 rpsL50 MC4100 DW12 relAI flbBS301 deoCI pfsF2S rbsR F- 480d/lacZAMJ5 endAI recAl hsdRJ7(rk-,mk+) supE44 thi-J DH5a gyrA relAI A(lacZYA-argF) U169 ATG1 A(lac-pro) supE thi hsdD5/F' tra36 proA+B+ IacP I-acZAMJ5 Plasmids pBR322 frdA+lB+C+D+ pH3 pH3 pH3FrdBC57S frdA+Bc57sC+D+ pH3 pH3FrdBC62S frdA+Bc62sC+D+ frdA+Bc6ssC+D+ pH3 pH3FrdBC65S pH3 pH3FrdBC77S frdA+Bc77sC+D+ Phage M13mp8 frdA'B+C' M13mp8 M13KW1
coworkers (30). Oligonucleotides for mutagenesis and sequencing were synthesized on a Biosearch model 8700. The oligonucleotides differ from the wild type by having one nucleotide altered, thus changing cysteine residues 57, 62, 65, and 77 to serine residues. The mutagenesis was performed using single-stranded M13KWI DNA as a template. The mutants were identified by DNA sequence analysis using the dideoxy termination proce ure of Sanger et al. (25) (Pharmacia sequencing kit). Strain TG1 was used for mutagenesis and single-stranded DNA sequence analysis. Following mutagenesis, the 2.0-kb EcoRI-Sal I fragment containing the frdB region was cloned back into the large EcoRI-Sal I fragment of plasmid pH3 to restore the complete frdABCl) operon. The mutation in this construct was confirmed by double-stranded DNA sequencing, using strain DH5a to prepare DNA. DNA was prepared using the Qiagen plasmid kit from Diagen (Chatsworth, CA). The four frdB mutant plasmids are designated pH3FrdBC57S, pH3FrdBC62S, pH3FrdBC65S, and pH3FrdBC77S (Table 1). Growth of Bacteria. For biochemical analysis the mutant plasmids were transformed into DW12 [A(frdABCD)]. The E. coli strains were grown anaerobically on glycerol/fumarate, glycerol/nitrate, and glucose/fumarate minimal media as described (6). Cells used for preparation of the membrane fraction and purified enzyme complexes were grown anaerobically on glucose/fumarate medium to stationary phase, chilled, and harvested by centrifugation. For phage and plasmid manipulations, cells were grown on Luria broth (31) and' solid media. Purification of Membranes Enriched in Fumarate Reductase. Sixty grams (wet weight) of E. coli DW12 containing the appropriate plasmid was suspended in 300 ml of 30 mM Tris/HCl (pH 8.0). The inner-membrane/fumarate reductase tubule fraction was then purified as described (32). The membranes obtained were enriched 2- to 3-fold for fumarate reductase, based on assay of covalent flavin {8a-[N(3)-histidyl] FAD where N(3) is Ni1, over the crude membrane preparations previously used (6). Enzyme Assays. Fumarate-dependent oxidation of reduced benzyl viologen (BVred) and menaquinol-6 (MQH2) and succinate-dependent reduction of phenazine methosulfate (PMS) were determined as described (6). EPR Spectroscopy. Samples for EPR spectroscopy were prepared under an argon atmosphere in a Vacuum Atmospheres (Hawthorne, CA) glove box (
