THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 275, No. 17, Issue of April 28, pp. 12400 –12409, 2000
`Printed in U.S.A.
`
`Site-directed Mutagenesis of 2,4-Dichlorophenoxyacetic
`Acid/a-Ketoglutarate Dioxygenase
`IDENTIFICATION OF RESIDUES INVOLVED IN METALLOCENTER FORMATION AND SUBSTRATE BINDING*
`
`(Received for publication, January 18, 2000)
`
`Deborah A. Hoganद, Sheila R. Smithi, Eric A. Saarii, John McCrackeni, and
`Robert P. Hausinger‡§**‡‡
`From the ‡Center for Microbial Ecology and Departments of §Microbiology, iChemistry, and **Biochemistry, Michigan
`State University, East Lansing, Michigan 48824
`
`acid (2,4-D)/a-ketoglut-
`2,4-Dichlorophenoxyacetic
`arate (a-KG) dioxygenase (TfdA) is an Fe(II)-dependent
`enzyme that catalyzes the first step in degradation of
`the herbicide 2,4-D. The active site structures of a small
`number of enzymes within the a-KG-dependent dioxyge-
`nase superfamily have been characterized and shown to
`have a similar HXDX50 –70HX10RXS arrangement of resi-
`dues that make up the binding sites for Fe(II) and a-KG.
`TfdA does not have obvious homology to the dioxygen-
`ases containing the above motif but is related in se-
`quence to eight other enzymes in the superfamily that
`form a distinct consensus sequence (HX(D/E)X138 –207
`HX10R/K). Variants of TfdA were created to examine the
`roles of putative metal-binding residues and the func-
`tions of the other seven histidines in this protein. The
`H167A, H200A, H213A, H245A, and H262A forms of TfdA
`formed inclusion bodies when overproduced in Esche-
`richia coli DH5a; however, these proteins were soluble
`when fused to the maltose-binding protein (MBP). MBP-
`TfdA exhibited kinetic parameters similar to the native
`enzyme. The H8A and H235A variants were catalytically
`similar to wild-type TfdA. MBP-H213A and H216A TfdA
`have elevated Km values for 2,4-D, and the former
`showed a decreased kcat, suggesting these residues may
`affect substrate binding or catalysis. The H113A, D115A,
`MBP-H167A, MBP-H200A, MBP-H245A and MBP-H262A
`variants of TfdA were inactive. Gel filtration analysis
`revealed that the latter two proteins were highly aggre-
`gated. The remaining four inactive variants were ex-
`amined in their Cu(II)-substituted forms by EPR and
`electron spin-echo envelope modulation (ESEEM) spec-
`troscopic methods. Changes in EPR spectra upon addi-
`tion of substrates indicated that copper was present at
`the active site in the H113A and D115A variants. ESEEM
`analysis revealed that two histidines are bound equato-
`rially to the copper in the D115A and MBP-H167A TfdA
`variants. The experimental data and sequence analysis
`lead us to conclude that His-113, Asp-115, and His-262
`
`are likely metal ligands in TfdA and that His-213 may aid
`in catalysis or binding of 2,4-D.
`
`2,4-Dichlorophenoxyacetic acid (2,4-D)1/a-ketoglutarate (a-
`KG) dioxygenase (TfdA) is an Fe(II)- and a-KG-dependent en-
`zyme that catalyzes the first step in degradation of the herbi-
`cide 2,4-D. This enzyme couples the oxidative decarboxylation
`of a-KG to the hydroxylation of a side chain carbon atom. The
`resultant hemiacetal spontaneously decomposes to form 2,4-
`dichlorophenol and glyoxalate (1). Mechanistically, TfdA re-
`sembles numerous other a-KG-dependent dioxygenases from
`plants, animals, fungi, and bacteria that catalyze similar hy-
`droxylation reactions at unactivated carbon centers (2, 3).
`Members of the a-KG-dependent dioxygenase superfamily
`are not closely related by their sequences but rather appear to
`fall into one of three groups of related enzymes or fall into a
`fourth group of unrelated sequences (4). The best studied a-KG-
`dependent hydroxylases, including prolyl and lysyl hydroxy-
`lase
`(5) and flavanone hydroxylase
`(3, 6), have an
`HXDX;55HX10RXS motif in common (7, 8), and this motif is
`present in the more than 20 enzymes, defined here as Group I,
`within the a-KG dependent dioxygenase superfamily (8, 9).
`Site-directed mutagenesis studies have confirmed the impor-
`tance of these residues for activity (8, 10 –13), and the crystal
`structures of two Group I enzymes, isopenicillin N synthase
`(IPNS) and deacetoxycephalosporin C synthase (DAOCS), in-
`dicate that these residues comprise the metallocenter and
`a-KG-binding site (14 –17). TfdA is not closely related in se-
`quence to the Group I a-KG-dependent dioxygenases described
`
`SCHEME 1
`
`* This research was supported in part by National Science Founda-
`tion Grants DEB9120006 and MCB 9603520 (to R. P. H), National
`Institutes of Health Grant GM 54065 (to J. M.), and the Michigan State
`University Agricultural Experiment Station. The costs of publication of
`this article were defrayed in part by the payment of page charges. This
`article must therefore be hereby marked “advertisement” in accordance
`with 18 U.S.C. Section 1734 solely to indicate this fact.
`¶ Supported in part by National Institutes of Health Biotechnology
`Training Grant T32-GM08350.
`‡‡ To whom correspondence should be addressed: 160 Giltner Hall,
`Dept. of Microbiology, Michigan State University, East Lansing, MI
`48824. Tel.: 517-353-9675; Fax.: 517-353-8957; E-mail: Hausinge@
`pilot.msu.edu.
`
`above but is clearly homologous (25–30% identity) to Esche-
`richia coli taurine/a-KG dioxygenase (TauD) (18) and sulfon-
`ate/a-KG dioxygenase from Saccharomyces cerevisiae (19).
`Furthermore, PSI-BLAST analyses (20) find additional rela-
`
`1 The abbreviations used are: 2,4-D, 2,4-dichlorophenoxyacetic acid;
`a-KG, a-ketoglutarate; TfdA, 2,4-D/a-KG dioxygenase; TauD, taurine/
`a-KG dioxygenase; cw-EPR, continuous wave electron paramagnetic
`resonance; ESEEM, electron spin echo envelope modulation; IPNS,
`isopenicillin N synthase; DAOCS, deacetoxycephalosporin synthase;
`MBP, maltose-binding protein; FT, Fourier transform; MOPS, 4-mor-
`pholinepropanesulfonic acid.
`
`This is an Open Access article under the CC BY license.
`
`12400
`
`This paper is available on line at http://www.jbc.org
`
`Inari Ex. 1019
`Inari Agric. v. Corteva Agriscience
`PGR2023-00022
`Page 00001
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`

`

`TfdA Mutagenesis and Metal Ligand Identification
`
`12401
`
`Group I
`(IPNS, DAOCS, and related enzymes)
`
`TABLE I
`Consensus motifs for subgroups within the a-KG-dependent dioxygenase superfamily
`X50–70
`
`HXD
`
`HX10(R/K)XS
`
`Group II
`(TfdA, TauD, clavaminate synthase and related enzymes)
`
`HX(D/E)
`
`X138–207
`
`HX10–13R
`
`Group III
`(Phytanoyl-CoA hydroxylase, proline hydroxylase and related enzymes)
`
`HXD
`
`X72–101
`
`HX10(R/K)XS
`
`tionships to g-butyrobetaine hydroxylase and clavaminate syn-
`thase. Alignment of these Group II sequences indicates the
`conservation of two histidines and one aspartate (His-113, His-
`262, and Asp 115 in TfdA) as well as an invariant arginine that
`may be analogous to the a-KG-binding arginine in DAOCS and
`related enzymes (Table I). A third set of enzyme sequences
`(Group III) from members of the a-KG-dependent dioxygenase
`superfamily, including phytanoyl-CoA hydroxylase and proline
`hydroxylase, exhibit the presence of a third related motif de-
`spite the lack of overall sequence similarity to Group I or Group
`II enzymes.
`In this study, we used site-directed mutagenesis methods to
`examine the roles of potential metal-binding residues in the
`above motif (His-113, His-262, and Asp-115) and the remaining
`seven histidines in TfdA. Previously published work showed
`that TfdA was inactivated by diethylpyrocarbonate, a histi-
`dine-selective reagent, and provided evidence consistent with
`the presence of multiple histidines in the active site (21). Spec-
`troscopic studies of TfdA showed the presence of two equatori-
`ally bound imidazole nitrogens as ligands to the active site
`metal and indicated that one imidazole ligand may be displaced
`or shifted to an axial position upon substrate binding (22–24).
`Based on analyses of different TfdA variants, we identify sev-
`eral likely metal ligands and provide evidence that another one
`or two histidines may aid in substrate binding.
`
`EXPERIMENTAL PROCEDURES
`Recombinant Plasmids—All plasmids were constructed from
`pUS311 (21), a pUC19 derivative that contains the Ralstonia eutropha
`JMP134 tfdA gene (Fig. 1). The H8A, D115A, H213A, H216A, H235A,
`H245A, and H262A TfdA variants were created by direct mutation of
`tfdA in pUS311 by the Stratagene Quickchange System (Stratagene, La
`Jolla, CA). All mutagenic primers are listed in Table II. Two alternative
`approaches were used to construct the three remaining variants. Plas-
`mids encoding H113A and H167A TfdA variants were created by
`CLONTECH mutagenesis of pXHtfdA, a pUC19 plasmid containing the
`59-XbaI-HindII fragment of the tfdA gene (Fig. 1). To create the com-
`plete gene containing the indicated mutations, the XbaI-HindII frag-
`ment was cloned into pHKtfdA, which contains the 39 end of the tfdA
`gene. pHKtfdA was constructed in two steps. First, the 1.4-kilobase pair
`XbaI-SalI fragment from pUS311, containing the complete tfdA gene,
`was cloned into pBC KS2 (Stratagene) cut with XbaI and XhoI to create
`pBCtfdA. This step had the benefit of eliminating a HindII site that
`interfered with further cloning steps. The 727-base pair HindII-KpnI
`fragment of pBCtfdA was subcloned into pBC KS2 cut with the same
`enzymes to give pHKtfdA. Similarly, the gene encoding H200A TfdA
`was made by mutagenesis of pXXtfdA, a pUC19 plasmid containing the
`59-XbaI-XhoI fragment of tfdA. The altered XbaI-XhoI fragment was
`then inserted into pHKtfdA cut with XbaI and XhoI to give the complete
`H200A tfdA gene. The identity of all final constructs was confirmed by
`sequence analysis. To insert the genes encoding H167A and H262A
`variants of TfdA into a plasmid that would allow for isopropyl-1-thio-
`b-D-galactopyranoside-controlled expression, the XbaI-SalI fragments
`from the corresponding plasmids described above were cloned into
`pET23a (Novagen) prepared with the same enzymes.
`To create the maltose-binding protein (MBP)-TfdA fusion proteins,
`the wild-type tfdA gene was amplified from pUS311 with TfdA-MBPF
`and TfdA-MBPR primers (Table II) to create an XbaI site directly
`upstream of the GTG start codon of the tfdA gene and a HindIII
`restriction site 54 base pairs downstream of the stop codon. The polym-
`erase chain reaction product was cloned directly into the pGEM-T
`vector according to the manufacturer’s instructions (Promega, Madison,
`
`FIG. 1. Restriction maps of the plasmids used in construction
`of tfdA mutants. The abbreviations for the restriction enzyme sites are
`as follows: H, HindII; X, XbaI; Xh, XhoI; Sl, SalI; K, KpnI; Sc, SacI; and
`N, NruI. The vector backbone is indicated in the box on the left. The tfdA
`open reading frame is shown as a wide, solid line.
`
`Mutant
`
`TABLE II
`Sequences of mutagenic primers used to create altered tfdA genes
`Primer sequencea
`59-CGC AAA TCC CCT TGC TCC TCT TTT CGC C-39
`H8A
`59-GTC GCT GGC CCA AGC TGG-39
`H113A
`59-GCA CAG CGC CAG CTC CTT TCA-39
`D115A
`59-GCG TGC CGA GCA GTA CGC ACT G-39
`H167A
`59-GGT TCG AAC CGC CGC CGG CTC-39
`H200A
`59-GCT CGC GGC CGC GCC GAT-39
`H213A
`59-CCT TCG ACG GCG CTC GCG-39
`H216A
`59-GCT TCT CGA GGC GAC ACA G-39
`H235A
`59-GTG TAC CGG GCT CGC TGG AAC-39
`H245A
`59-CGT TCT TGC ACG CGG ACG CAG-39
`H262A
`Tfda-MBPF 59-TCT CTA GAG TGA GCG TCG TCG CAA ATC C-39
`Tfda-MBPR 59-GTC AAG CTT GGT TGC GTA CAT CTT GTG G-39
`a The reverse primer used for the creation of all mutants was the
`complement of the forward primer.
`
`WI). The XbaI-HindIII fragment was isolated from the resulting plas-
`mid and cloned into the pMAL-c2 vector that had been digested with the
`same enzymes. The identity of the newly created malE-tfdA gene fusion
`was confirmed by sequencing. Substitution of the mutation-containing
`internal NruI fragment for the same fragment of the wild-type malE-
`tfdA gene created MBP-fusion forms of altered TfdAs. First, pMAL-tfdA
`was digested with NruI, and the vector fragment was purified and
`religated to create pMAL-tfdADNruI. The resultant plasmid was lin-
`earized with NruI and dephosphorylated with calf intestine alkaline
`phosphatase prior to ligation with the NruI fragments isolated from the
`previously described mutant genes. Constructs were confirmed by re-
`striction analysis.
`Protein Purification—H8A, H113A, D115A, H216A, H235A, and
`wild-type TfdA proteins were purified from E. coli DH5a cells carrying
`pUS311 and its mutated derivatives according to a previously described
`protocol (21). In addition, the non-mutated enzyme and the TfdA vari-
`ants H167A, H200A, H213A, H245A, and H262A were purified as
`MBP-TfdA fusion proteins from E. coli DH5a by the protocol described
`in the pMAL Protein Fusion and Purification System Manual (New
`England Biolabs, Beverly, MA).
`Analysis of Kinetic Parameters—Specific activities of the wild-type
`and variant TfdA proteins were determined by a previously described
`spectrophotometric assay (21). The typical assay mixture contained 1
`mM 2,4-D, 1 mM a-KG, 100 mM (NH4)2Fe(SO4)2, and 100 mM ascorbic acid
`in 10 mM MOPS buffer (pH 6.75) at 30 °C. The reactions were quenched
`by the addition of EDTA to a concentration of 5 mM. 2,4-Dichlorophenol
`was quantified by reaction with 4-aminoantipyrene followed by meas-
`urement of the absorbance at 510 nm. One unit of activity was defined
`as
`the amount
`of
`enzyme
`required to produce 1 mmol
`of
`dichlorophenolzmin21. Protein concentrations were determined using
`
`PGR2023-00022 Page 00002
`
`

`

`12402
`
`TfdA Mutagenesis and Metal Ligand Identification
`
`the Bio-Rad Protein Assay with bovine serum albumin as a standard.
`For calculation of the kcat values, the TfdA variants were assumed to
`have Mr 5 31,600 and the MBP-TfdA variants were assigned Mr 5
`74,500.
`The low Km values for a-KG (;2–5 mM for the wild-type enzyme)
`precluded use of the 4-aminoantipyrene assay for accurate determina-
`tion of this value. The alternative method used to measure the Km
`values for a-KG quantified the amount of 14CO2 liberated from a-[1-
`14C]KG during the course of the reaction (21).
`Native Protein Analysis by Gel Filtration—Size exclusion chromatog-
`raphy was used to estimate the native molecular weights of TfdA,
`MBP-TfdA, and mutant proteins. The proteins were chromatographed
`on a Superose 6 gel filtration column (1.0 3 30 cm, Amersham Phar-
`macia Biotech) in 20 mM Tris buffer (pH 7.5), 1 mM EDTA, and 200 mM
`NaCl at a flow rate of 0.2 mlzmin21. The elution volumes were compared
`with those for gel filtration standards (Bio-Rad) including thyroglobu-
`lin, 670 kDa; bovine gamma globulin, 158 kDa; chicken ovalbumin, 44
`kDa; myoglobin, 17 kDa; and vitamin B12, 1350 Da.
`Spectroscopic Analysis—Proteins for electron paramagnetic reso-
`nance (EPR) and electron spin-echo envelope modulation (ESEEM)
`spectroscopic analyses were exchanged into 25 mM MOPS (pH 6.75) by
`repeated concentration and dilution in Centricon 30 (Amicon) centrifu-
`gal concentrators. The final subunit concentration was 0.5 mM for the
`non-fusion forms of TfdA and 0.4 mM for the MBP-TfdA proteins. CuCl2
`was added to a concentration of 450 and 350 mM, respectively. Buffered
`solutions of a-KG and 2,4-D were added to final concentrations of 5 mM.
`Glycerol was present at 40% in all samples.
`X-band EPR spectra were obtained at 77 K on a Bruker ESP-300E
`spectrometer. ESEEM data were collected on a home-built spectrome-
`ter; the microwave bridge of this instrument has been previously de-
`scribed in detail (25). Data collection and analyses were controlled by a
`Power Computing model 200 Power PC using software written with
`LabView version 5.01 (National Instruments). Electron spin echoes
`were digitized, averaged, and integrated by a Tektronix model 620B
`digital oscilloscope interfaced to the spectrometer computer via an
`IEEE-488 bus. Two four-channel delay and gate generators (Stanford
`Research Systems model DG535), a Bruker BH-15 magnetic field con-
`troller, and a Hewlett-Packard model 8656B radiofrequency synthe-
`sizer were also interfaced using IEEE-488 protocol. Data were collected
`using a reflection cavity that employed a folded microstrip resonator
`(26). A three-pulse stimulated echo sequence (90-t-90°-T-90°) was used.
`ESEEM spectra were generated by Fourier transformation of the time
`domain data using dead time reconstruction (27). Simulations of the
`experimental data were performed on a Sun SparcII work station.
`Simulation programs were written in FORTRAN and based on the
`density matrix formalism developed by Mims (28). Software for the
`frequency analysis of the experimental and simulated data was written
`in Matlab (Mathworks, Natick, MA).
`Sequence Comparisons—Related sequences were initially detected
`by BLAST (29) and PSI-BLAST (20) analyses. Alignments were gener-
`ated with the CLUSTAL algorithm (30), and the figure was prepared
`using Genedoc (31).
`
`RESULTS
`Production of the Mutant TfdAs—Initially, all of the mutant
`genes were expressed from their pUC19-based plasmids except
`for those encoding H113A, H167A, and H200A TfdA, which
`were in pBC KS2-derived plasmids. By using the standard
`protocol to produce soluble, wild-type TfdA (growth at 30 °C to
`early stationary phase), only the H8A, H113A, D115A, H216A,
`and H235A variants existed as soluble proteins. All of the other
`TfdA variants were present as inclusion bodies even when
`grown at lower temperatures (22 °C), in M9 minimal medium,
`or in LB broth containing 660 mM sorbitol and 2.5 mM betaine
`(32). In addition, isopropyl-1-thio-b-D-galactopyranoside-con-
`trolled production of H167A and H262A proteins from mutant
`genes cloned into pET23a did not yield soluble samples even
`when the harvested cell pellets were suspended in buffer con-
`taining 20% glycerol to limit protein aggregation.
`To overcome the solubility problems for the five TfdA vari-
`ants, MBP-TfdA fusion proteins were created. Wild-type TfdA
`and the MBP-TfdA fusion protein had essentially identical kcat
`and very similar Km values for a-KG and 2,4-D (Table III). A
`slight increase in the apparent KD for Fe(II) may reflect some
`
`TABLE III
`Summary of the kinetic parameters for active TfdA variants
`
`TfdA sample
`
`kcat
`
`Km aKG
`
`Km 2,4-D
`
`KD Fe(II)
`
`min21
`mM
`mM
`mM
`2.0 6 0.5
`19.3 6 3.7
`4.9 6 0.73
`442 6 33
`Wild type
`15.2 6 5.4
`33.7 6 2.5
`9.9 6 1.8
`411 6 23
`MBP fusion
`1.9 6 0.7
`17.7 6 3.8
`6.5 6 1.8
`284 6 6.9
`H8Aa
`27.7 6 9.8
`318 6 44
`8.3 6 2.9
`22.1 6 2.9
`MBP-H213A
`2.4 6 1.7
`52 6 5
`6.05 6 2.6
`474 6 47
`H216A
`1.4 6 1.7
`34 6 8
`9.6 6 1.6
`284 6 25
`H235A
`a More than 75% of the protein was present as the degradation
`product. The kcat was estimated from the active, full-length fraction.
`
`metal binding capacity of MBP. Since the presence of the fusion
`protein did not appear to greatly affect the kinetic parameters
`of wild-type enzyme, similar fusion proteins were created for
`the H167A, H200A, H213A, H245A, and H262A TfdA variants.
`Kinetic Analyses of Altered TfdAs—Results from kinetic
`analyses of the four active mutant proteins are summarized in
`Table III. H8A TfdA was soluble and active but was rapidly
`proteolyzed to an inactive form. By electrophoretic compari-
`sons, the cleavage site appeared to be the same as in wild-type
`TfdA (between Arg-77 and Phe-78) (21). The rate of proteolysis
`of H8A TfdA was enhanced compared with that seen for the
`wild-type enzyme despite the presence of EDTA and protease
`inhibitors in the purification buffer. Because purified H8A
`TfdA was more than 75% degraded, the catalytic rate constant
`was calculated with the estimated amount of intact enzyme.
`These calculations indicate rates and Km values similar to
`those for the wild-type enzyme. Similarly, the kinetic parame-
`ters for H235A TfdA were comparable to the native enzyme. In
`contrast, two variants exhibited differences from wild-type en-
`zyme in their kinetic parameters. The H213A MBP-TfdA var-
`iant exhibited a 20-fold reduction in kcat and a 10-fold increase
`in Km for 2,4-D. In addition, H216A TfdA had a modest (2.5-
`fold) increase in the Km for 2,4-D and no change in catalytic
`rate. The other kinetic parameters for H213A MBP-TfdA and
`H216A TfdA (Km for a-KG and KD for ferrous ion) did not differ
`significantly from the wild-type values. Six soluble TfdA vari-
`ants (H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A,
`and MBP-H262A) exhibited no activity even when assayed
`with elevated substrate and cofactor concentrations (10 mM
`a-KG, 5 mM 2,4-D, and 250 mM Fe(II)).
`Evaluation of the Structural Consequences of the Muta-
`tions—To assess whether the inactive mutant proteins as-
`sumed conformations similar to the wild-type enzyme, their
`apparent molecular weights were estimated by gel filtration
`analysis. The observed size of wild-type TfdA was found to be
`51 kDa by comparison to protein standards, suggesting that
`TfdA forms a compact dimer or an elongated monomer. The
`elution volume for both H113A and D115A corresponded ex-
`actly to wild-type TfdA indicating that these proteins are not
`significantly altered in their quaternary structure. MBP-TfdA
`eluted both in the void volume (approximately 25% of the
`protein) and at a position corresponding to 216 kDa (roughly
`75% of the protein), suggesting that MBP-TfdA forms at least a
`dimer. Because each MBP-TfdA subunit is comprised of two
`domains separated by a 13-amino acid linker, the resultant
`protein may migrate with a larger apparent molecular weight.
`MBP-H167A and MBP-H200A samples demonstrated the same
`two-peak profile as MBP-TfdA but with larger proportions elut-
`ing in the void volume. MBP-H245A and MBP-H262A proteins
`were soluble; however, gel filtration analysis indicated the
`presence of only highly aggregated material eluting in the void
`volume. Because these mutant proteins exhibited aberrations
`in their folding properties, the catalytic role of His-245 and
`His-262, if any, could not be assessed.
`
`PGR2023-00022 Page 00003
`
`

`

`TfdA Mutagenesis and Metal Ligand Identification
`
`12403
`
`EPR Spectroscopic Characterization of Variants with Altered
`Metal Sites—The metallocenter properties for selected TfdA
`variants were probed by EPR spectroscopy. To circumvent the
`problems that arise in EPR measurements of integer spin para-
`magnetic centers, Fe(II) was substituted with cupric ion. Al-
`though the Cu(II) form of TfdA is inactive, Cu(II) binds com-
`petitively with respect to Fe(II) (Ki 5 1–3 mM), and copper-
`substituted TfdA has been used previously to study the metal
`coordination environment of this enzyme in the presence and
`absence of substrates (22–24). Spectral parameters of wild-type
`Cu(II)-TfdA, Cu(II)-TfdA 1 a-KG, and Cu(II)-TfdA 1 a-KG 1
`2,4-D (Fig. 2A and Table IV), agreed well with those reported
`previously (23, 24). Earlier studies of copper-substituted wild-
`type TfdA indicated that the metal is bound in a type 2 envi-
`ronment with a mixture of O and N ligands in the equatorial
`plane. Upon addition of a-KG and 2,4-D to the enzyme, the
`spectral parameters are altered to a more rhombic signal with
`accompanying resolution of ligand hyperfine coupling (Fig. 2A).
`These results suggest that binding of the co-substrates to the
`enzyme leads to a better defined copper site with a-KG binding
`directly to the metallocenter (23, 24). The small Ai (less than 14
`mT) for the a-KG- and 2,4-D-bound sample indicates a signif-
`icant distortion from planarity.
`The four inactive mutant forms of TfdA with quaternary
`structures similar to the corresponding wild-type protein
`(H113A, D115A, MBP-H167A, and MBP-H200A) were ana-
`lyzed by EPR spectroscopy to assess the metal coordination
`environments. No significant differences between the spectra of
`MBP-TfdA and the non-fusion wild-type TfdA were observed
`(data not shown). EPR spectra of the copper-substituted sam-
`ples, in all cases, showed contributions from multiple copper
`sites indicating a mixture of copper centers, most likely result-
`ing from copper binding in multiple conformations or at sites
`other than the active site. The presence of alternative copper-
`binding sites is not surprising in an enzyme with nine histidines.
`The EPR spectra for Cu-H113A TfdA alone and in the pres-
`ence of a-KG and 2,4-D (Fig. 2B and Table IV) differ signifi-
`cantly from spectra of wild-type enzyme and show modest
`changes in Cu(II) g values and hyperfine tensor principal val-
`ues upon substrate additions. The broadening of the EPR sig-
`nal in the gi region upon addition of a-KG again suggests a
`mixture of copper site conformations. Thus, it appears that
`alteration of His-113 significantly affects the metal binding
`properties for TfdA such that copper is no longer constrained to
`a single active site configuration.
`The EPR spectrum of D115A-TfdA (Fig. 2C) has parameters
`similar to those observed in the copper-substituted wild-type
`protein (Table IV), although with less resolution of Ai. Addition
`of a-KG (in the presence or absence of 2,4-D) has a dramatic
`effect on the appearance of the D115A data, enhancing resolu-
`tion of the Cu(II) hyperfine peaks at gi and the ligand hyperfine
`structure in the g’ region (Fig. 2C). The appearance of these
`superhyperfine interactions may result from a subtle shift in
`the orientation of the principal g tensor such that the imidazole
`ligands occupy an increasingly equatorial position (33). These
`results are consistent with the formation of a tighter or more
`regular copper-binding site upon addition of the co-substrate.
`The EPR spectrum for MBP-H167A TfdA (Fig. 2D), like that
`for the H113A variant, is poorly resolved, probably due to
`binding of copper in multiple configurations instead of forma-
`tion of one major conformation. Additionally, few significant
`changes are seen upon addition of either a-KG or 2,4-D. The
`MBP-H200A EPR spectrum (Fig. 2E) is better resolved than
`that of H113A or MBP-H167A and clearly shows a second set of
`resonances of lower amplitude with parameters identical to
`those observed for the copper-substituted wild-type enzyme
`
`FIG. 2. X-band CW-EPR spectra of Cu(II)-substituted TfdA
`variants. EPR spectra were obtained at 77 K for the following samples
`(substrate concentrations were 5 mM, when present). Top to bottom,
`wild-type Cu-TfdA in the absence of substrates, with a-KG, and with
`a-KG 1 2,4-D (A). H113A Cu-TfdA in the absence of substrates, with
`a-KG, and with a-KG 1 2,4-D (B). D115A Cu-TfdA in the absence of
`substrates, with a-KG, and with a-KG 1 2,4-D (C). MBP-H167A Cu-
`TfdA in the absence of substrates, with a-KG, and with a-KG 1 2,4-D
`(D). MBP-H200A Cu-TfdA in the absence of substrates and in the
`presence of a-KG 1 2,4-D (E). Spectral parameters are listed in Table
`IV.
`
`(Table IV). The presence of this wild-type signal suggests that
`His-200 is most likely not a copper-binding ligand in TfdA. As
`found for H113A and MBP-H167A, addition of a-KG and/or
`2,4-D has little effect on the spectrum.
`ESEEM Spectroscopic Characterization of Variants with Al-
`tered Metal Sites—Pulsed EPR (ESEEM) spectroscopy has
`proven useful for determining the number of histidines bound
`to copper in proteins. This approach has been previously used
`to determine that copper-substituted wild-type TfdA binds cop-
`
`PGR2023-00022 Page 00004
`
`

`

`12404
`
`TfdA Mutagenesis and Metal Ligand Identification
`
`TABLE IV
`Summary of the EPR spectral parameters for
`Cu(II)-substituted TfdA variants
`
`Wild-type TfdA
`Wild-type TfdA/a-KG
`Wild-type TfdA/a-KG/2,4-D
`H113A
`H113A/a-KG
`H113A/a-KG/2,4-D
`D115A
`D115A/a-KG
`
`D115A/a-KG/2,4-D
`
`MBP-H167A
`MBP-H167A/a-KG/2,4-D
`MBP-H200A
`
`MBP-H200A/a-KG/2,4-D
`
`g\
`
`2.34
`2.36
`2.38
`2.30
`2.33
`2.35
`2.30
`2.30
`2.34
`2.30
`2.34
`2.30
`2.30
`2.30
`2.34
`2.30
`2.34
`
`A\
`
`mT
`16.3
`15.1
`12.0
`16.6
`15.8
`14.0
`17.0
`16.2
`16.8
`16.2
`16.8
`17.1
`17.1
`17.1
`16.1
`17.1
`16.1
`
`g’
`
`2.07
`2.07
`2.09
`2.07
`2.07
`2.08
`2.06
`2.06
`2.06
`2.06
`2.06
`2.07
`2.07
`2.07
`2.07
`2.07
`2.07
`
`per in a site with two histidyl residues directly coordinated to
`the metal. As previously reported (23), three-pulse ESEEM
`spectra collected in the gi region for wild-type TfdA exhibit
`sharp peaks at 0.6, 0.9, and 1.5 MHz and a broad feature at 3.5
`MHz (Fig. 3B). This signature is typical of imidazole bound in
`an equatorial position to copper. The additional appearance of
`narrow combination bands at 2.1, 2.5, and 3.1 MHz in these
`spectra indicated the presence of at least two such imidazole
`ligands bound to the copper (34 –36). Spectral simulations us-
`ing the density matrix approach of Mims (28) were used to
`analyze these data further including determination of the num-
`ber of histidyl ligands coordinated to copper. The gi ESEEM of
`Cu(II)-substituted wild-type TfdA was simulated with mag-
`netic parameters for copper equatorially coordinated to two
`identical histidines with a simulation program using the angle
`selection scheme developed for ENDOR analysis (37, 38). For
`each simulation, a background decay function was applied to
`allow the amplitudes of the initial 1.5 ms of the simulations to
`match the data. The resulting simulated data sets (dashed
`curves) are superimposed on the experimental data in Fig. 3.
`Further analysis of these data suggested that formation of the
`ternary complex with a-KG and 2,4-D results in the probable
`loss or reorientation of one of these imidazole ligands, as evi-
`denced by a substantial decrease in the modulation intensity
`and disappearance of the combination bands (23).
`Three-pulse ESEEM patterns for the H113A (trace A), MBP-
`H167A (trace B), and D115A (trace C) variants of TfdA are
`shown in Fig. 4. Each data set was collected under identical
`conditions and normalized so that the integrated echo ampli-
`tudes range from zero, determined by shifting the integration
`window off of the signal at the end of a scan, to one which
`marks the largest measured echo amplitude. Low frequency
`modulations indicative of equatorially bound histidyl ligand(s)
`are observed for all four mutant TfdAs. The ESEEM from
`H113A (trace A) and MBP-H200A (not shown) variants are
`nearly identical and show modulations that are considerably
`weaker than those measured for MBP-H167A (trace B) and
`D115A (trace C). Each of these data contains a large unmodu-
`lated or DC component (well over 80% of the total signal inten-
`sity in the case of H113A), contrasting sharply with the
`ESEEM for copper-substituted wild-type TfdA, where the DC
`component constituted less than 40% of the total signal inten-
`sity (Fig. 3A).
`The frequency spectra, obtained by Fourier transformation
`(FT) of the data shown in Fig. 4, are characterized by major
`
`FIG. 3. Three-pulse ESEEM spectrum and Fourier transforma-
`tion of Cu(II)-substituted wild-type TfdA with 2-histidine simu-
`lation data. ESEEM spectrum of wild-type Cu(II)-TfdA 1 a-KG (solid)
`and computer-simulated data for Cu(II) bound by two identical histi-
`dines (dashed) (A) and the corresponding frequency domain spectrum
`(B). Parameters for data collections were magnetic field 5 2800 G, t 5
`375 ns, T 5 50 ns, n5 8.98 GHz; 4.2 K. Cu(II) hyperfine parameters: gxy
`5 2.05, gz 5 2.34, Axx 5 Ayy 5 20 MHz, Azz 5 487 MHz; Superhyperfine
`parameters: gN 5 0.40347, Aiso 5 1.7 MHz, radius 5 2.75 Å [u,f] 5
`[2.37,2.07] radians, e2qQ 5 1.60 MHz, h 5 0.75, NQI [a,b,g] 5
`[0.50,1.1,0.1] radians. The simulated data were treated with an expo-
`nential decay function to give yij 5 (yij 20.17)zexp((t 1 T)/3500)1.5) 1
`0.17. For each simulation, a background decay function was applied to
`allow the amplitudes of the initial 1.5 ms of the simulations to match the
`data.
`
`FIG. 4. Three-pulse ESEEM time domain spectra of TfdA vari-
`ants. H113A Cu-TfdA (A), magnetic field 5 3050 G, t 5 300 ns, T 5 40
`ns. MBP-H167A Cu-TfdA (B), magnetic field 5 3100 G, t 5 300 ns, T 5
`40 ns. D115A Cu-TfdA (C), magnetic field 5 3050 G, t 5 385 ns, T 5 55
`ns, n 5 8.8 GHz; 4.2 K.
`
`features at 0.7, 1.5, and 4.0 MHz that are indicative of histidyl
`imidazole equatorially coordinated to Cu(II) (36). The spectra
`obtained for D115A and MBP-H167A variants are shown in
`Figs. 5B and 6B (solid lines), respectively. The resolution in
`these spectra is poor when compared with similar data ob-
`tained for rigid Cu(II) proteins or model complexes (39, 40). In
`Fig. 5A, the normalized three-pulse ESEEM data for D115A
`TfdA (solid line) are shown with the computer-simulated data
`
`PGR2023-00022 Page 00005
`
`

`

`TfdA Mutagenesis and Metal Ligand Identification
`
`12405
`
`FIG. 5. Spectral simulation of ESEEM data for D115A-Cu-TfdA.
`Experimental (solid line), computer-simulated 1-His (dashed line), and
`simulated 2-His (dotted line) time domain spectra (A) and the corre-
`sponding Fourier-transformed data (B) for copper-substituted D115A
`TfdA are shown. Magnetic field 5 3050 G, t 5 230 ns, T 5 50 ns G, n 5
`8.8 GHz, 4.2 K, gN 5 0.40347, e2qQ 5 1.55 MHz, h 5 0.87, Axx 5 1.6
`MHz, Ayy 5 1.9 MHz, Azz 5 2.2 MHz. The simulated data were treated
`with an exponential decay function to give yjj 5 (yjj 20.05)zexp((t 1
`T)/600)0.8) 2 0.05.
`
`FIG. 6. Spectral simulation of ESEEM data for MBP-H167A-Cu-
`TfdA. Experimental (solid line), computer-simulated 1-His (dashed
`line), and 2-His (dotted line)