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J. Biol. Chem., Vol. 279, Issue 19, 19977-19986, May 7, 2004

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Probing the Active Site Loop Motif of Murine Ferrochelatase by Random Mutagenesis^*

Zhen Shi and Gloria C. Ferreira¶||

From the Department of Biochemistry and Molecular Biology, College of Medicine and ^¶H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612

Received for publication, December 17, 2003 , and in revised form, January 30, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction. Functional substitutions in the 10 consecutive loop positions Gln²⁴⁸–Leu²⁵⁷ were identified by genetic complementation in Escherichia coli strain vis. Lys²⁵⁰, Val²⁵¹, Pro²⁵³, Val²⁵⁴, and Pro²⁵⁵ tolerated a variety of replacements including single substitutions and contained low informational content. Gln²⁴⁸, Ser²⁴⁹, Gly²⁵², Trp²⁵⁶, and Leu²⁵⁷ possessed high informational content, since permissible replacements were limited and only observed in multiply substituted mutants. Selected active loop variants exhibited k_cat values comparable with or higher than that of wild-type murine ferrochelatase. The K_m values for porphyrin increased, except for the single mutant V251L. Other than a moderate increase observed in the triple mutant S249A/K250Q/V251C, the K_m values for Fe²⁺ were lowered. The k_cat/K_m for porphyrin remained largely unchanged, with the exception of a 10-fold reduction in the triple mutant K250M/V251L/W256Y. The k_cat/K_m for Fe²⁺ was improved. Molecular modeling of these active loop variants indicated that loop mutations resulted in alterations of the active site architecture. However, despite the plasticity of the loop primary structure, the relative spatial positioning of the loop in the active site appeared to be maintained in functional variants, supporting a role for the loop in ferrochelatase function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ferrochelatase (EC 4.99.1.1 [EC] ; protoheme ferrolyase), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. Ferrochelatase has been identified in a large number of organisms (reviewed in Refs. 1–3). In eukaryotes, ferrochelatase is nucleus-encoded with an average of 400–500 amino acid residues and associated with the inner mitochondrial membrane. Prokaryotic ferrochelatases are smaller, and whereas most are associated with the plasma membrane, some are soluble (reviewed in Ref. 3). [2Fe-2S] clusters have been identified in animal ferrochelatases (4, 5), in the yeast Schizosaccharomyces pombe (6), and in the bacterial species Caulobacter crescentus and Mycobacterium tuberculosis (7), but their roles remain elusive. Genetic defects in mammalian ferrochelatase cause a metabolic disease, erythropoietic protoporphyria (8, 9).

The x-ray crystal structures of ferrochelatases from Bacillus subtilis (10, 11), human (12), and yeast Saccharomyces cerevisiae (13) reveal that they share similar folding patterns and active site structures. Whereas B. subtilis ferrochelatase is a monomer (10, 11), human (12) and yeast (13) ferrochelatases are homodimers. In all cases, each monomeric unit consists of two domains, each of which is folded into a Rossmann-type fold with a four-stranded, parallel -sheet flanked by -helices on both sides (10–13). Molecular dynamics calculations of the B. subtilis ferrochelatase with bound nickel-protoporphyrin (14) and the crystal structure of the ferrochelatase-N-methyl mesoporphyrin complex (11) indicate that porphyrin binds to a deep cleft between the two domains. This cleft is enriched with many conserved residues important for catalysis (10–12). One side of the active site cleft is formed by the first two N-terminal -helices, ₁ and ₂, and a loop between them, and the other side consists of a short loop sequence, which connects a -strand and an -helix in the second domain (10–12). Although the overall sequence identity is 20%, this loop motif is conserved among all ferrochelatases (Fig. 1).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Alignment of the amino acid sequences of ferrochelatase in the loop region. A representative subset illustrates the high degree of homology in the loop motif among all known sequences of ferrochelatase. The amino acid sequences were obtained from NCBI using BLAST search and aligned using ClustalW (47).

In order to examine the role of the conserved loop motif in ferrochelatase function, we used random mutagenesis and genetic complementation to identify functional substitutions and to evaluate the information content of each of the 10 consecutive loop residues in murine ferrochelatase. We found that the ferrochelatase loop motif possesses a considerable degree of plasticity, tolerating multiple substitutions within the motif, albeit the overall spatial arrangement of the loop was maintained in functional variants. Kinetic characterization of selected, active variants further substantiated the importance of the loop in ferrochelatase function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Oligonucleotides were synthesized by Cybersyn. Restriction enzymes and DNA polymerases were from New England Biolabs. Hemin, protoporphyrin IX, and zinc-protoporphyrin were from Frontier Scientific. MOPS,¹ Tween 80, Tween 20, bovine serum albumin, the bicinchoninic acid protein determination reagents, CelLytic B II bacterial cell lysis extraction reagent, ExtrAvidin-Peroxidase, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, and N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethyl-ammonium salt (biotin-DHPE) were from Sigma. Phosphatidylcholine (bovine liver), phosphatidylethanolamine (bovine liver), phosphatidylinositol (bovine liver), phosphatidylserine (porcine brain), lysophosphatidylcholine (soy), lysophosphatidyl-ethanolamine (chicken egg), cardiolipin (bovine heart), cholesterol, diacylglycerol, and sphingomyelin (porcine brain) were from Avanti Polar Lipids. A T7 sequenase version 2.0 DNA sequencing kit, ECL chemiluminescent Western blotting detection reagents, and Hyperfilm ECL were from Amersham Biosciences. Plasmid mini kits were from Qiagen. TALON metal affinity resins and a TALON HT 96-well plate were from BD Biosciences. Blue Sepharose CL-6B, PD-10 columns, and Microspin S200-HR columns were from Amersham Biosciences. Amicon stirred cells and Amicon Ultra-4 centrifugal filter units were from Millipore Corp. Protran BA85 nitrocellulose membrane (0.45 µm) was from Schleicher and Schuell.

Plasmids and Bacteria—Plasmid pGF47 contains the N-terminal His-tagged, mature form of wild-type murine ferrochelatase (14). Overexpression of ferrochelatase was performed as previously described (15). Plasmid pGF23 contains the murine, erythroid 5-aminolevulinate synthase cDNA cloned into the pCASS3 vector (16). Escherichia coli strain vis was a kind gift of Dr. H. Inokuchi at Kyoto University (17).

Construction of a Dummy Vector—Plasmid pZS1 was generated by replacing the segment in the murine ferrochelatase expression plasmid (pGF47) spanning the 10 codons for the loop motif with a stuffer sequence derived from pGF23 via two unique restriction sites, BstEII and PspOMI.

Construction of the Ferrochelatase Random Library—The ferrochelatase random library was constructed based on previously described protocols (18–20). Two single-stranded DNA oligonucleotides with 15 complementary base pairs at their 3'-ends were annealed. Oligonucleotide 1 (5'-ATG GAA AAG CTG GGT TAC CCC AAC CCC TAC CGA CTG GTT TGG-3') is a 42-mer corresponding to the sense strand nucleotides and contains a BstEII site (italic type) for cloning. Oligonucleotide 2 (5'-AGC GTC ATC TGT CTG AGG GCC CAA CCA GGG TAC TGG ACC AAC CTT GGA CTG CCA AAC CAG TCG GTA-3') is a 66-mer spanning the antisense strand nucleotides with a PspOMI restriction site (italic type) for cloning. Oligonucleotide 2 contains degenerate nucleotides (underlined) corresponding to codons 248–257 ² of the mature murine ferrochelatase loop residues. Each of the 10 loop codons was randomized using 85% wild-type nucleotides and a 15% mixture of the other three nucleotides. A 60-µl annealing reaction, containing 0.5 nmol of each oligonucleotide, 200 mM Tris-HCl, pH 7.5, 100 mM MgCl₂, and 250 mM NaCl was incubated at 80 °C for 5 min, followed by 55 °C for 15 min, 37 °C for 15 min, and finally room temperature for 30 min. The hybrid was extended using the Klenow fragment of E. coli DNA polymerase I (5 units) in a 40-µl mixture containing 50 pmol of annealed oligonucleotides, a 62.5 µM concentration of each of the four dNTPs, and EcoPol buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, and 7.5 mM dithiothreitol). The extension reaction was carried out at 37 °C for 2 h. The generated double-stranded mutagenic DNA was then amplified by PCR using two primers corresponding to the 5' termini of oligonucleotides 1 and 2. The two primers used were as follows: FC-66, 5'-ATG GAA AAG CTG GGT TAC CCC AA-3', which covers the first 23 nucleotides of the 5' terminus of oligonucleotide 1 and FC-R67, and 5'-AGC GTC ATC TGT CTG AGG-3', which corresponds to the 18 nucleotides of the 5' terminus of oligonucleotide 2. The PCRs were performed in 100-µl reaction volumes consisting of 20 pmol of primers FC-66 and FC-R67, 3–50 pmol of the extended double-stranded DNA as template, 50 µM each of the four dNTPs, 1 unit of Vent_R DNA polymerase, and ThermoPol reaction buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, and 0.1% Triton X-100). The reaction mixture was subjected to temperature cycling in a programmable thermal controller (MJ Research) by running 1 cycle of 95 °C for 2 min, followed by 30 cycles of 95 °C for 1 min, 45 °C for 2 min, and 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR product of 93 bp was purified using Microspin S200-HR columns (Amersham Biosciences), and subsequently digested with BstEII and PspOMI. The digested, 60-bp fragment was isolated from an agarose gel using GLASSFOG matrix and buffers in the MERmaid kit (Q-BIOgene) and subcloned into the dummy vector pZS1 previously digested with the same enzymes. The 20-µl ligation reaction contained the randomly mutagenized insert and vector at a 5:1 molar ratio, 1 mM ATP, 7 units of T4 DNA ligase (New England Biolabs), ligase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol, 1 mM ATP, and 25 µg/ml bovine serum albumin), 100 mM NaCl, and 10% polyethylene glycol 6000. After 16–20 h at 16 °C, the ligation reaction was purified by passing through Microspin S200-HR columns (Amersham Biosciences) and used to transform electrocompetent vis cells with an Electroporator II (Invitrogen). Aliquots of 1–2 µl of ligated products were electroporated into 40 µlof vis cells, using 0.1-cm cuvettes (Invitrogen) at 1.5 kV, 50 microfarads, and 150 ohms. After the pulse, transformants were resuspended in 1 ml of SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, and 20 mM glucose) supplemented with 10 µg/ml hemin and incubated at 37 °C for 4 h with continuous shaking. To assess transformation efficiency, a small portion of the transformed cells was spread onto LB plates containing 50 µg/ml ampicillin, 0.4% (w/v) glucose and 10 µg/ml hemin. The remaining transformed cells were plated onto LB/ampicillin plates to allow selection of the active ferrochelatase variants.

Genetic Selection in E. coli and DNA Sequence Analysis of the Functional Variants—Only vis cells expressing functional ferrochelatase can form colonies on hemin-free media. Aliquots of vis cells transformed with the random library ligation products were plated on LB-agar medium containing 50 µg/ml ampicillin and 0.4% (w/v) glucose and incubated at 37 °C for 24–30 h. Plasmids were isolated from all of the surviving colonies, and the randomized regions were sequenced to determine the permissible amino acid substitutions. To prepare double-stranded DNA templates, 20 ml of vis culture for each clone was grown overnight in Terrific Broth (21) containing 50 µg/ml ampicillin and 0.4% (w/v) glucose, and plasmids were purified using Qiagen Tip-20 (Qiagen). Sequences from 166 clones were determined by the dideoxynucleotide chain termination method (22) using a T7 sequenase 2.0 DNA sequencing kit (Amersham Biosciences) and [³⁵S]dATP (PerkinElmer Life Sciences). The remaining 48 clones were sequenced by the ABI automated DNA sequencers at the University of Florida.

Zinc Chelatase Activity Assay of the Functional Variants—vis cells containing the active random variants were grown overnight at 37 °Cin Terrific Broth (21) containing 50 µg/ml ampicillin and 0.4% (w/v) glucose. These cultures were used to inoculate fresh medium at a 1:20 dilution; cells were grown at 37 °C to an A₆₀₀ value of 0.8–1.0. 3 ml of vis cell cultures were pelleted, and crude cell extracts were prepared to a final volume of 1.2 ml, using the CelLytic B II bacterial cell lysis extraction reagent according to the manufacturer's instructions (Sigma). Zinc chelatase activity was determined by a fluorescence assay, using a RF-5301PC fluorimeter equipped with a red-sensitive photomultiplier tube (Shimadzu). Briefly, 300 µl of the cell extract was mixed with 100 mM Tris-HCl, pH 7.6, 3 µM ZnCl₂, 2 µM protoporphyrin IX solution in a final volume of 2 ml. The reaction mixture was incubated at 37 °C for 1 h and the zinc-protoporphyrin formed was measured fluorimetrically by determining its emission at 592 nm using an _ex = 421 nm (23). Zinc chelatase activity was calculated as nmol of zinc-protoporphyrin produced per min by converting the rate of change in fluorescence intensity (arbitrary units min^-1), using a standard curve of fluorescence intensity versus zinc-protoporphyrin concentration.

Purification of Wild-type and Mutant Ferrochelatase—The wild-type murine ferrochelatase and selected, functional loop variants were expressed under the E. coli alkaline phosphatase promoter phoA as described previously (15) and purified as N-terminal His-tagged proteins. The corresponding plasmids were transformed into DH5 or BL21(DE3) cells, and the bacteria were grown overnight at 30 °C in MOPS medium containing 50 mg/liter ampicillin to promote protein overexpression (15). Wild-type ferrochelatase and single mutant V251L were purified using blue Sepharose affinity column chromatography according to an established protocol (15).

The remaining four functional loop variants, including single mutant P255R, triple mutants S249A/K250Q/V251C and K250M/V251L/W256Y, and quadruple mutant Q248P/S249G/K250P/G252W, were purified using Talon metal-chelating affinity column chromatography followed by gel filtration chromatography. 8-Liter cultures of bacterial cells harboring the ferrochelatase variants were pelleted and then resuspended in 20 mM Tris-HCl, pH 8, containing 10% glycerol. After cell rupture by a French press (3–4 passages at 14,000 p.s.i.), the homogenate obtained was further solubilized in 20 mM Tris-HCl, pH 8, containing 0.5 M NaCl and 0.5% cholate under constant stirring at 4 °C for 30 min and subsequently ultracentrifuged at 44,000 rpm for 1 h. The supernatant was loaded onto a gravity column packed with 10 ml of Talon resin (BD Biosciences) and previously equilibrated with 20 mM Tris-HCl, pH 8, containing 10% glycerol, 150 mM NaCl, and 0.5% cholate (equilibration buffer). The resin was first washed with equilibration buffer, followed by sequential washes of equilibration buffer supplemented with increasing concentrations of imidazole stepping from 10 to 20 to 30 mM. Ferrochelatase was eluted from the Talon column with the equilibration buffer containing 100 mM imidazole. The eluted ferrochelatase was concentrated in an Amicon stirred cell and stored in liquid N₂. For use in kinetic analyses, aliquots of concentrated ferrochelatase were loaded onto a PD-10 column (Amersham Biosciences) and eluted in 20 mM Tris-HCl, pH 8, containing 10% glycerol, 150 mM NaCl, and 0.5% cholate.

Protein purity was assessed by SDS-PAGE (24), and protein concentration was determined using the bicinchonic acid assay with bovine serum albumin as a standard. UV-visible absorption spectra of purified ferrochelatase and variants were recorded using an UVPC-2100U dual beam spectrophotometer (Shimadzu) equipped with a 1-cm path length quartz cuvette at a controlled temperature of 23 °C. Determination of the metal ion content of S249A/K250Q/V251C and wild-type ferrochelatase using plasma emission spectroscopy was performed by the Chemical Analysis Laboratory at the University of Georgia.

Steady-state Kinetic Analyses—Ferrochelatase activity was determined by monitoring the consumption of the protoporphyrin substrate using ferrous iron as the metal substrate in a continuous spectrofluorimetric assay conducted under strictly anaerobic conditions as previously described (25). Typically, a mixture containing 100 mM Tris acetate, pH 8.1, 0.5% (v/v) Tween 80, protoporphyrin IX was incubated with ferrochelatase for 5 min at 30 °C, and the enzymatic reaction was initiated by ferrous ammonium citrate injection (25). Activities are expressed as nmol of protoporphyrin consumed per min per mg of ferrochelatase. Steady-state kinetic parameters were determined from matrices of five protoporphyrin and five Fe²⁺ concentrations. The reported values and the S.D. values were obtained from the nonlinear least squares fit of the data to the Michaelis-Menten equation for bireactant systems using the software DataFit (Oakdale Engineering) (25).

Liposome Binding Assay: Ferrochelatase (or Loop Variant)-Lipid Interactions—All of the functional variants in the library were purified using a TALON HT 96-well plate (BD Biosciences). The equilibration, wash, and elution buffers were the same as for purification by Talon affinity column. Eluate from each well was concentrated, and imidazole was removed by buffer exchange using an Amicon Ultra-4 centrifugal filter unit (Millipore). Liposomes were formulated with a lipid composition similar to that found in the inner mitochondrial membranes of mouse liver cells (26). Biotinylated liposomes were prepared following a standard procedure (27). Each lipid was weighed and suspended in chloroform to make a 1 mg/ml stock solution. The stocks were mixed at ratios (w/w) of 1% biotin-DHPE, 35% phosphatidylcholine, 26.5% phosphatidylethanolamine, 18% cardiolipin, 5% phosphatidylinositol, 2.3% cholesterol, 2.8% diacylglycerol, 0.7% sphingomyelin, 0.5% phosphatidylserine, 0.5% lysophosphatidylcholine, 0.3% lysophosphatidylethanolamine, 2.2% palmitic acid, 1.2% stearic acid, 1.3% oleic acid, 1.8% linoleic acid, and 0.9% arachidonic acid. The mixture was dried under N₂; resuspended in 25 mM Tris, pH 7.6, and 150 mM NaCl (TBS); and sonicated to obtain a liposome solution at 100 µg/ml. Liposome size distribution was determined at 26 °C by dynamic light scattering using a submicron particle size analyzer (Beckman Coulter). For the protein-lipid interaction assay, a dilution series of each purified variant was spotted onto a nitrocellulose membrane (Schleicher and Schuell), blocked in 50 ml of Superblock buffer (Pierce) for 2 h at room temperature (23 °C), and probed with 50 µl of liposome stock solution diluted in 50 ml of TBS at 30 °C for 1 h. After washing three times in TBS with 0.05% (v/v) Tween 20 (TBST), the blot was incubated with peroxidase-conjugated extravidin (Sigma) at a 1:50,000 dilution for 30 min at room temperature. Following extensive washing, the blot was incubated in chemiluminescence substrate solutions (Amersham Biosciences) according to the manufacturer's instructions and exposed to Hyperfilm (Amersham Biosciences). The bound liposomes were quantified using the densitometry program supplied with the ChemiImager 4400 (Alpha Innotech). The relative binding affinity was calculated from the intensity of the chemiluminescent signals plotted against the amount of protein spotted onto the nitrocellulose membrane. The wild-type ferrochelatase was run as a standard, and a variant (G252D/V254I) was used as an internal control; essentially, G252D/V254I was included in every single blot to ensure that the ratio between the chemiluminescent signal intensity for the wild-type ferrochelatase and G252D/V254I remained constant from blot to blot.

Molecular Modeling of Mouse Ferrochelatase—Comparative protein modeling of the three-dimensional structures of the wild-type murine ferrochelatase and selected loop variants was performed using the amino acid sequence and coordinates for the human ferrochelatase (Protein Data Bank accession code 1hrk [PDB] ) as the template. Sequence alignments, molecular modeling, energy minimization, and model analyses using PROCHECK were performed on the Geno3D servers provided by the Institute of Biology and Chemistry of Proteins (Lyon, France) (28). The monomeric models for wild-type ferrochelatase and the loop variants were visualized using the program VMD version 1.7 (29). The secondary structures in the protein models were identified by the STRIDE algorithm included in VMD (29).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Construction of the Random Sequence Library and Selection of Active Ferrochelatase Variants—To assess the role of the conserved loop motif in ferrochelatase function, particularly with respect to interaction with the porphyrin substrate, random sequence mutagenesis was employed to analyze the information content of each loop residue. Random substitutions at the 10 consecutive positions Gln²⁴⁸–Leu²⁵⁷ in murine ferrochelatase were introduced by incorporating a mixture of all four nucleotides at each of the 30 bases. Using a mixture of 85% of the wild-type nucleotides and 15% of the other nucleotides at each position, the bias was set toward the wild-type sequence, thus increasing the probability of recovering active ferrochelatase variants. Under these conditions, a full spectrum of permissible substitutions at each position was drawn. Two major factors were critical in the construction of the random sequence library. First, a mock or inactive vector was constructed as the cloning vector of the random nucleotide sequences. Since the mock vector could not express a functional ferrochelatase variant, the selection of active, random ferrochelatase variants was not affected by the background activity derived from the vector. Second, E. coli strain vis was chosen as the host for the plasmid library containing the randomized loop sequences, because it allows positive genetic complementation for identification of the active variants. Since E. coli vis cells need hemin in growth media for survival (17), transformants of vis cells carrying the mock vector with inactivated ferrochelatase could not grow in hemin-free media, whereas vis cells transformed with a functional ferrochelatase expression plasmid could override the requirement for hemin and form colonies on LB/ampicillin plates.

Design of the Random Library—Screening of the unselected library by genetic complementation showed that 90% of the random mutants were inactive. This is consistent with the hypothesis that the loop motif is important for enzymatic function and also demonstrates that the biological selection procedure was efficient.

To calculate the expected rate of mutation, the probability for substitution to occur at a specific residue is given by _x = 1 - (1 - r) (1 - r) (1 - r/3), where r is the probability that one particular nucleotide is replaced by any of the three remaining bases, assuming that there are, on average, three codons for each amino acid (20). In our random library, r = 0.15, and _x = 1 - (1 - 0.15)² x (1 - 0.15/3) = 0.314. The frequencies of detecting single mutations at specific positions were calculated. For instance, the likelihood of generating P253T alteration (CCN to CAN) would be P_PT = (r/3) x (1 - r) = (0.15/3) x (1 - 0.15) = 0.0425, and the probability of obtaining a clone with the only mutation P253T is (1 - 0.314)⁹ x (0.0425) = 0.00143. From screening a total of 2,210 clones, four single mutants of P253T were recovered in the active library, corresponding to a mutation rate of 0.0018, very similar to the calculated value.

Evaluation of Selected, Active Ferrochelatase Variants— From a total of 2,210 vis transformants harboring randomized loop mutations, 214 clones were functional as identified by their ability to grow in hemin-free media, and the plasmids were sequenced to detect nucleotide changes in the loop motif. As shown in Fig. 2A, the active variants were predominantly single, double, and triple mutants, with an average of 2.2 amino acid changes. No wild-type ferrochelatase was recovered in this collection. For a relatively quick assessment of the active library, all of the functional, vis transformants were assayed for enzymatic activity by monitoring zinc-protoporphyrin production (23) using crude cell extracts. Most variants showed reduced zinc chelatase activity to <60% of the wild-type level, whereas a subset of triple mutants displayed activities comparable with or even higher than wild-type ferrochelatase (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Distribution of amino acid substitutions in functional loop variants. A, distribution of the functional substitutions relative to the number of amino acid changes. Random mutations were introduced into the 10 residues encoding the murine ferrochelatase loop motif. Active variants were recovered by genetic complementation in E. coli strain vis, and mutations were identified by DNA sequencing. B, zinc chelatase activity relative to the number of amino acid substitutions. Zinc chelatase activity was assayed by monitoring zinc-protoporphyrin formation using cell extracts prepared from vis cells transformed with the active variants (see "Experimental Procedures"). The zinc chelatase activity in each variant was calculated relative to wild-type ferrochelatase.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Spectrum and frequency of amino acid substitutions in functional loop variants. Mutations at each residue are listed along with the number of times each substitution was observed. The wild-type loop sequence is shown on the bottom of each mutant category.

View larger version (73K): [in this window] [in a new window]   FIG. 4. Interaction between functional ferrochelatase loop variants and membrane lipids. A, polarity distribution at each loop position in the functional ferrochelatase variants. Basic amino acid substitutions accumulated in C-terminal positions Gly252, Pro253, Val254, Pro255, and Leu257. Hydrophobic residues include Ala, Phe, Gly, Ile, Leu, Met, Pro, Val, and Trp; polar residues include Cys, Asn, Gln, Ser, Thr, and Tyr; acidic residues include Asp and Glu; basic residues include His, Lys, and Arg. B, protein-lipid interactions among wild-type ferrochelatase and functional loop variants. Proteins were purified in a 96-well format, spotted onto a nitrocellulose membrane in a dilution series, and probed with biotinylated liposomes (see "Experimental Procedures"). Bound liposomes were detected using peroxidase-conjugated extravidin and visualized using the ECL system. Examples are shown for increased binding with the variant P253Q/V254K/P255T (row 1) and reduced binding with the variant Q248P/S249G/K250P/G252W (row 2) when compared with the wild-type ferrochelatase (row 3); variant G252D/V254I (row 4) was included as an internal control (see "Experimental Procedures"). C, analysis of the liposomal binding affinities of the loop variants. The right panel illustrates the liposome-binding affinities of each active variant normalized against that of the wild-type ferrochelatase (see "Experimental Procedures"). The left panel indicates the polarity of individual loop residues in each variant using a color code as follows: blue for hydrophobic residues, yellow for polar residues, black for acidic residues, and red for basic residues. Mutated positions in each variant are marked by an X.

As previously determined for mature, wild-type murine ferrochelatase (15), SDS-polyacrylamide gel electrophoresis analysis of the purified active loop variants yielded a subunit molecular mass of 42 kDa (data not shown). The UV-visible absorbance spectra of the purified proteins indicated that, similar to the wild-type protein, most variants contained [2Fe-2S] clusters, characterized by an absorption band at 330 nm and shoulders at 440 and 525 nm (Fig. 5) and with absorption ratios A₃₃₀/A₂₇₈ and A₄₄₀/A₂₇₈ identical to those determined for the wild-type ferrochelatase (30). Curiously, unlike the wild-type protein, the triple mutant S249A/K250Q/V251C lacked the prominent shoulder region around 440 nm (Fig. 5), and metal content analysis by plasma emission spectroscopy indicated that the ratio of iron/protein was 50% higher than that of the wild-type enzyme (data not shown), which has a stoichiometry of 1 [2Fe-2S] cluster per monomer.

View larger version (19K): [in this window] [in a new window]   FIG. 5. UV-visible absorbance spectra of the purified wild-type murine ferrochelatase and selected active loop variants. Recombinant wild-type ferrochelatase and variants were overproduced in E. coli DH5 or BL21(DE3) cells and purified using blue Sepharose or metal chelate affinity column chromatography (see "Experimental Procedures"). The absorption spectra are shown for wild-type ferrochelatase (8.8 µM) (a), single mutant V251L (15.2 µM) (b), single mutant P255R (16.8 µM) (c), triple mutant S249A/K250Q/V251C (18.8 µM) (d), triple mutant K250M/V251L/W256Y (10.5 µM) (e), and quadruple mutant Q248P/S249G/K250P/G252W (19.4 µM)(f). Similar to the wild-type ferrochelatase, four variants exhibited the electronic absorption features characteristic of [2Fe-2S] cluster-containing proteins, including absorption bands at 330 nm and shoulders at 440 and 525 nm. The triple mutant S249A/K250Q/V251C lacked the absorption shoulder at 440 nm.

View larger version (61K): [in this window] [in a new window]   FIG. 6. Molecular modeling of wild-type murine ferrochelatase and selected loop variants (monomeric). The structural models were generated by homology modeling on Geno3D servers (28), using human ferrochelatase (Protein Data Bank code 1hrk [PDB] ) as the template. Substituted residues in the loop motif (red) are marked (green). The color scheme for the secondary structures is as follows: -helix in blue, -strand in yellow, 310-helix in lime green, -helix in purple, and turns and coils in cyan. The N-terminal 2-helix and its extension in the loop variants are indicated by open arrows; the second domain -helix and its variations are indicated by filled arrows. When not occluded by structural elements, the N and C termini are indicated. The three-dimensional models are shown for wild-type murine ferrochelatase (A), single mutant V251L (B), single mutant P255R (C), triple mutant S249A/K250Q/V251C (D), triple mutant K250M/V251L/W256Y (E), and quadruple mutant Q248P/S249G/K250P/G252W (F). Images were generated using VMD version 1.7 (29).

For all loop variants, changes in secondary structure were observed in the N-terminal domain forming one of the sides of the active site cleft and in the second domain -helix region, located adjacent to the active site cleft (Fig. 6). At the N terminus, immediately following the second -helix (₂), the variants showed an increase of helical content in place of the relatively unstructured turns. Whereas in the structural model of the wild-type protein, the ₂-helix consists of 10 residues, Gln⁵²–Lys⁶¹ (Fig. 6A), in the loop variants, the ₂-helix was extended by a short -helix or 3₁₀-helix to include 5–7 additional residues (Fig. 6, B–F). These additional residues along with the meandering loop that follows appear to form contact with residues in the second domain of the enzyme close to the active site cavity.

The structural model of wild-type murine ferrochelatase revealed a short -helix, consisting of five residues ²⁹⁰TLYEL²⁹⁴ in the second domain near the bottom of the active site pocket (Fig. 6A), in an analogous fashion to the -helix observed in the crystal structures of human (³⁴⁹ELDIE³⁵³) and yeast (³¹⁸EIDLG³²²) ferrochelatases (35). This -helix was replaced by an -helix in the triple mutant S249A/K250Q/V251C (Fig. 6D). In contrast, the quadruple mutant Q248P/S249G/K250P/G252W had a long, acidic residue-enriched -helix corresponding to the ²⁸⁹ETLYELDIEY²⁹⁸ sequence (Fig. 6F). In the single mutants V251L and P255R and the triple mutant K250M/V251L/W256Y, the -helix shifted positions slightly to include two glutamate residues (Fig. 6, B, C, and E). The -helix was formed by five residues: ²⁹³ELDIE²⁹⁷ in V251L and P255R and ²⁸⁹ETLYE²⁹³ in K250M/V251L/W256Y.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The observation that the ferrochelatase loop motif is in close proximity to the bound porphyrin macrocycle (11, 14) prompted us to assess the role of the conserved loop motif in ferrochelatase function, particularly in porphyrin binding. In the work reported here, we applied random mutagenesis to generate substitutions within the target loop sequence of murine ferrochelatase (Gln²⁴⁸–Leu²⁵⁷) and demonstrated that whereas most positions in the loop can accommodate functionally permissible substitutions, mutations of specific loop residues disrupt the interaction between the porphyrin substrate and ferrochelatase.

Informational Content Analysis—Despite the conservation of the ferrochelatase loop motif (Fig. 1), every amino acid within this sequence tolerated functionally permissible substitutions (Fig. 3). The degree of acceptable substitutions varied, however, from position to position, with the wild-type murine ferrochelatase residues Lys²⁵⁰, Val²⁵¹, Pro²⁵³, Val²⁵⁴, and Pro²⁵⁵ occupying low informational content positions. Some of the single amino acid substitutions identified at the low informational content positions were also co-selected in the multiply substituted variants (Fig. 3), and in fact, a few of these amino acid substitutions are also present in nature (Fig. 1). The remaining five positions in the loop, Gln²⁴⁸, Ser²⁴⁹, Gly²⁵², Trp²⁵⁶, and Leu²⁵⁷, had high informational content, since amino acid substitutions were only possible when they were associated with changes in other loop residues (Fig. 3). Whereas Gly²⁵² was the most amenable to change, even if drastic (e.g. substitution of Gly²⁵² with Trp or Arg) (Fig. 3), substitutions at Gln²⁴⁸, Ser²⁴⁹, and Trp²⁵⁶ were scarce, occurring primarily in triple, occasionally in quadruple and quintuple mutants, suggesting that complementary changes in the loop are necessary to counterbalance the loss of these wild-type residues. The high informational content of Trp²⁵⁶ agrees with a previous model in which hydrophobic and aromatic residues mediate porphyrin binding and distortion and thus are critical in ferrochelatase function (23).

Membrane Lipid Interaction—Membrane attachment of ferrochelatase may facilitate uptake of the water-insoluble porphyrin substrates from a hydrophobic environment and also provide a pathway for heme release (12, 36). Indeed, the crystal structures of human and yeast ferrochelatases demonstrated that the active site entrance is delimited by two, oppositely located loops, which are rich in hydrophobic residues (12, 36) and were previously hypothesized to facilitate membrane association (12, 36). Further, a few positively charged residues in these loops were suggested to interact with the phosphate groups of the membrane phospholipids (13). One of these loops surrounding the active site entrance corresponds to the murine ferrochelatase sequence Gln²⁴⁸–Leu²⁵⁷, and consistent with the above hypothesis, we observed an accumulation of positively charged amino acid substitutions at the C-terminal hydrophobic loop residues Gly²⁵², Pro²⁵³, Val²⁵⁴, and Pro²⁵⁵ (Fig. 4A). They conferred an overall increase in the binding affinities of the variants toward liposomes mimicking the composition of inner mitochondrial membranes (Fig. 4C).

Effect of Loop Motif Mutations on Ferrochelatase Activity— An important step in ferrochelatase catalysis is the binding and distortion of porphyrin macrocycle to facilitate metal chelation (14, 37, 38). The increase in the values of the active loop variants (Table I) substantiates the proposal that the loop residues contribute to porphyrin-ferrochelatase interaction. K250M/V251L/W256Y was the only variant with both a lower k_cat and a higher than those of the wild-type ferrochelatase (Table I). Indeed, this variant has a catalytic efficiency toward porphyrin 10-fold lower than wild-type enzyme (Table I). Although in the K250M/V251L/W256Y variant 3 residues were mutated, we believe that the major effect was due to the W256Y mutation. Specifically, the V251L mutation alone did not bring a decrease in the k_cat value or a significant increase in the value of the enzyme (Table I), and mutation of the Lys²⁵⁰ residue should not affect dramatically the function of ferrochelatase, since this residue occupies a low information content position (Fig. 3). In addition, mutation of the corresponding residue in yeast ferrochelatase (Trp²⁸²) yielded a variant (W282L) exhibiting a 10-fold decrease in and an unaltered K_m for the metal substrate (32). Actually, the homologous residue in B. subtilis ferrochelatase (Trp²³⁰) was shown to stack against the pyrrole ring, thereby stabilizing the position of the porphyrin ring in the crystal structure (11).

The over 7-fold increase in the of the multiply mutated variant Q248P/S249G/K250P/G252W (Table I) probably stemmed from the G252W mutation. The replacement of Gly²⁵² with the bulky aromatic tryptophan possibly blocked the active site opening, thus hindering the entry and binding of the porphyrin substrate. This hypothesis agrees with the observation, based on the crystal structure of B. subtilis ferrochelatase, that any side chain other than glycine would introduce steric hindrance at this position (11). The triply mutated variant S249A/K250Q/V251C also exhibited a considerable increase (5-fold) in the . Whereas Ser²⁴⁹ contains high informational content (Fig. 3) and is buried deeply in the active site cavity, the Val²⁵¹ side chain is directed toward the path of porphyrin entry. Thus, it is possible that this combination of changes in the triple variant leads to misalignment or destabilization of porphyrin binding. Unexpectedly, with the exception of the triply mutated variant K250M/V251L/W256Y, k_cat increased in all of the loop variants (Table I). The increase, ranging from 2- to 4.4-fold, conceivably resulted from conformational flexibility of the loop motif. Surface loops are frequently identified as mobile elements mediating protein conformational changes (39–41). They may control access to the active site by adopting an open conformation to permit substrate entry and product release and a closed conformation to protect the active site from the exterior and promote enzyme-substrate interactions required for catalysis (39). We suspect that some of the loop variants adopted closed conformations more conducive to catalysis. For example, in the Q248P/S249G/K250P/G252W variant, the indole ring and the pyrrolidine rings at the 252- and 248/250-positions, respectively, might restrict the conformational plasticity of the loop region and promote a closed conformation more favorable to catalysis. Alternatively, a higher k_cat might result from stabilization of the reaction intermediates.

Although the iron substrate ligands in ferrochelatase remain to be unequivocally identified, nitrogenous and/or oxygenous ligands appear to coordinate the metal substrate (32, 42, 43). Active site His²⁰⁹ and Glu²⁸⁹ (murine ferrochelatase numbering) have been recognized as metal ligands and/or catalytically essential residues (11, 32, 33). More recently, the analysis of the co-crystal structures of yeast ferrochelatase with bound metal ions suggested that Ser²⁷⁵ with His²³⁵ and Glu³¹⁴ (equivalent to murine Ser²⁴⁹, His²⁰⁹, and Glu³¹⁴, respectively) ligate Co²⁺ and Cd²⁺ (13). Thus, an increase in the of the triple mutant S249A/K250Q/V251C may account for the loss of the stabilization provided by Ser²⁴⁹ (Table I). Curiously, the differences in the values of the loop variants correlate with the presence of a -helix in the second domain of ferrochelatase (Fig. 6). In a number of proteins, -helix residues function to coordinate metal ions required for enzymatic activity (35, 44, 45). In the structural model of murine ferrochelatase, the residues arranged along the helical edge of the -helix form a channel that connects the interior of the active site to the protein exterior (Fig. 6). The quadruple variant Q248P/S249G/K250P/G252W, whose value was the lowest among the analyzed variants (Table I), exhibits a long (i.e. 10 residues) -helix (Fig. 6F). Given that the unit rise per residue of a -helix is shortest among all helical types (10, 35), this alignment of the residues allows the side chains of Glu²⁸⁹, Glu²⁹³, and Glu²⁹⁷ to be more closely packed and possibly provides for a more efficient metal uptake; this might explain the decreased value of the quadruple variant. In contrast, S249A/K250Q/V251C is the only variant with a regular -helix in place of the -helix (Fig. 6D); this should result in a longer trajectory between the glutamate residues and thus slower metal transfer, which might account for the increase in its value. The mode of metal substrate interaction proposed here is consistent with the recent studies of metal binding in yeast ferrochelatase (13, 46). In this model, metal binding requires the conserved His²⁰⁹ (murine numbering) and occurs on the same side of the active site as that of porphyrin binding, whereas the distal residues, including glutamates in the -helical region, play a regulatory role by promoting metal release from the primary binding site (46).

In conclusion, a mutational survey of the putative porphyrin-binding loop, previously identified by structural analysis, has begun to reveal the role of the loop in substrate binding and catalysis of ferrochelatase. Whereas multiple functional substitutions were tolerated within the loop motif, the positions occupied by Gln²⁴⁸, Ser²⁴⁹, Gly²⁵², Trp²⁵⁶, and Leu²⁵⁷ exhibited the highest stringency. Kinetic analysis of selected, active variants suggested that the loop mutations result in a general disruption of the interaction between the porphyrin substrate and ferrochelatase. Resonance Raman spectroscopic studies are under way to provide a detailed characterization of the involvement of the loop residues in porphyrin distortion and ferrochelatase catalysis.

	FOOTNOTES

 
^* This work was supported by American Cancer Society Grant RSG-96-05106-TBE and American Heart Association/Florida Affiliate Grant 0051240B. 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.

Recipient of an American Heart Association/Florida Division Predoctoral Fellowship.

^|| To whom correspondence should be addressed. Tel.: 813-974-5797; Fax: 813-974-0504; E-mail: gferreir{at}hsc.usf.edu.

¹ The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; PPIX, protoporphyrin IX; TBS, Tris-buffered saline; biotin-DHPE, N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt.

² The numbering of amino acids in murine, mature ferrochelatase is according to the recent sequencing results in this laboratory and the mouse genome sequence submitted by the Genome Exploration Research Group in RIKEN, Japan (GenBank^TM nucleotide accession number AK004718 [GenBank] .2). In the previously deposited sequence (GenBank^TM protein accession number AAA80530 [GenBank] , two codons (i.e. for Gln⁶⁸ and Tyr⁶⁹) were omitted.

	ACKNOWLEDGMENTS

 
We thank Yujiang Song and Dr. J. Shelnutt at the University of New Mexico for taking the dynamic light scattering measurement of the liposome vesicles.

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