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Keywords:

  • manganese;
  • redox;
  • catalase;
  • spectroscopy;
  • X-ray structure

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

X-ray crystallography of the nonheme manganese catalase from Lactobacillus plantarum (LPC) [Barynin, V.V., Whittaker, M.M., Antonyuk, S.V., Lamzin, V.S., Harrison, P.M., Artymiuk, P.J. & Whittaker, J.W. (2001) Structure9, 725–738] has revealed the structure of the dimanganese redox cluster together with its protein environment. The oxidized [Mn(III)Mn(III)] cluster is bridged by two solvent molecules (oxo and hydroxo, respectively) together with a µ1,3 bridging glutamate carboxylate and is embedded in a web of hydrogen bonds involving an outer sphere tyrosine residue (Tyr42). A novel homologous expression system has been developed for production of active recombinant LPC and Tyr42 has been replaced by phenylalanine using site-directed mutagenesis. Spectroscopic and structural studies indicate that disruption of the hydrogen-bonded web significantly perturbs the active site in Y42F LPC, breaking one of the solvent bridges and generating an ‘open’ form of the dimanganese cluster. Two of the metal ligands adopt alternate conformations in the crystal structure, both conformers having a broken solvent bridge in the dimanganese core. The oxidized Y42F LPC exhibits strong optical absorption characteristic of high spin Mn(III) in low symmetry and lower coordination number. MCD and EPR measurements provide complementary information defining a ferromagnetically coupled electronic ground state for a cluster containing a single solvent bridge, in contrast to the diamagnetic ground state found for the native cluster containing a pair of solvent bridges. Y42F LPC has less than 5% of the catalase activity and much higher Km for H2O2 (≈1.4 m) at neutral pH than WT LPC, although the activity is slightly restored at high pH where the cluster is converted to a diamagnetic form. These studies provide new insight into the contribution of the outer sphere tyrosine to the stability of the dimanganese cluster and the role of the solvent bridges in catalysis by dimanganese catalases.

Abbreviations
LPC

Lactobacillus plantarum manganese catalase

TTC

Thermus thermophilus manganese catalase

LT-MCD

low temperature magnetic circular dichroism

MCD

magnetic circular dichroism

MPD

2-methyl-2,4-pentanediol

ABS

optical absorption

Catalases (E.C. 1.11.1.6) are antioxidant defence enzymes that catalyze the redox disproportionation of the toxic oxygen metabolite, hydrogen peroxide, into dioxygen and water [1]. Two distinct families of catalases are known, differing in both the architecture of the folded protein and in the nature of the catalytic redox cofactor, heme iron or nonheme manganese. While the heme-containing catalase family has been well characterized both structurally [2,3] and biochemically [4], the alternative, nonheme dimanganese catalases or ‘pseudocatalases’ are less extensively studied, although they appear to be widespread among prokaryotes. Manganese catalases have been isolated from bacteria (Thermus thermophilus[5], Thermoleophilum album[6], and Lactobacillus plantarum[7,8]) and a hyperthermophilic archeon (Pyrobaculum caldifontis[9]). X-ray crystal structures have been reported recently for the enzymes from T. thermophilus (TTC) [10] and L. plantarum (LPC) [11]. Both enzymes are hexamers of identical subunits organized around a catalytic core of close-packed four-helix bundle domains. Each of these coiled-coil domains binds two manganese ions forming novel redox-active binuclear manganese complexes that serve as the catalytic active sites.

The dimanganese complexes of LPC and TTC are templated by their environment in the protein, being bound by five amino acid side chains arising within the four-helix bundles (Fig. 1). A µ1,3-bridging glutamate carboxylate (Glu66, LPC numbering) anchors the two ions in the binuclear cluster (Fig. 2). Each Mn ion is further coordinated by one histidine (His69 to Mn1 and His181 to Mn2) and one glutamate (Glu35 to Mn1 and Glu148 to Mn2) bound to opposite faces of the cluster, and the manganese core is completed by two solvent-derived µ1,1 oxygen atom bridges. Analysis of the metal-ligand bond distances and hydrogen-bonding patterns in the crystalline complex shows that the two solvent bridges are structurally distinct, one (W2, trans to the coordinated histidine imidazoles) occurring as a protonated (aquo/hydroxo) group in the oxidized (3,3) state of the cluster, while the other (W1, trans to the pair of coordinated carboxylates) occurs as an unprotonated oxo ion (O2–). The glutamate coordinated to the Mn2 subsite is bidentate in LPC, chelating the metal center, while the Mn1 subsite has a monodentate glutamate ligand which allows a third, terminal solvent molecule to bind in the apical position. A nonligating glutamate Glu178 is poised above the cluster, forming a hydrogen bond to the apical water.

image

Figure 1. Subunit structure for Lactobacillus plantarum manganese catalase. The ribbon diagram (left) illustrates the organization of a single subunit from the hexameric protein. Secondary structural elements are color-coded: α-helix (magenta); β-sheet (yellow). The dimanganese catalytic core (cyan) is embedded in the center of a coiled-coil α-helical domain. (Right) Stereoview of dimanganese active site embedded in the interior of the four-helix bundle catalytic domain. (Based on PDB ID 1JKU). Rendered using Midasplus[12].

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image

Figure 2. The active site of Lactobacillus plantarum manganese catalase. Manganese ions (Mn1, Mn2) and coordinated solvent molecules (W1, W2, W3) are labeled. Dashed lines define the hydrogen bond network surrounding the active site (Based on PDB ID 1JKU). The structure was rendered using ortep-3 [13].

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The binuclear active site is embedded in a web of hydrogen bonds that radiate from the metal cluster into the outer sphere protein environment. Arg147 anchors a network of hydrogen bonds on the side trans to the coordinated histidines, with the guanidinium NH2 group bound to Glu148 and the bridging hydroxide (W2), while the guanidinium NH1 is hydrogen bonded to Glu35. In addition, the phenolic hydroxyl of Tyr42 is a hydrogen bond acceptor to the NE of Arg147 and a donor to Glu148. The arginine residue is unique to LPC but the same hydrogen bonding pattern involving tyrosine and a glutamate ligand is observed in TTC [10] and corresponding residues can be identified in a number of sequence homologs [11], suggesting that this interaction is a conserved feature of manganese catalase structures.

This conserved outer sphere interaction between a tyrosine residue and a glutamate ligand is particularly interesting in the context of related motifs that are important functional elements in other, more distantly related enzymes [14]. For example, the E. coli Class I ribonucleotide reductase (RNR) is based on a similar four-helix bundle domain architecture templating a binuclear iron (rather than manganese) active site complex. The outer sphere of this redox active metal center includes a tyrosine residue (Y122) that forms a catalytically essential free radical [15]. Likewise, the manganese cluster of the photosynthetic oxygen evolving complex is associated with a redox-active outer sphere tyrosine (YZ) which is involved in electron transfer and hydrogen atom abstraction steps of the oxygen synthesis reactions [16,17]. While there is no evidence for involvement of free radicals in manganese catalase turnover, the conservation of the outer sphere tyrosine within this family of enzymes [10,11], as well as the occurrence of a redox-active outer-sphere tyrosine in the heme catalases [18], suggests that this residue may make essential contributions to the stability or reactivity of the manganese active site. Attempts to express active manganese catalase in E. coli have been unsuccessful [19]. Development of a novel homologous expression system for recombinant manganese catalase (using a catalase-negative strain of L. plantarum) has made possible the dissection of the active site structure and function by site-directed mutagenesis. In this study we describe the detailed spectroscopic, biochemical and structural characterization of recombinant Y42F L. plantarum manganese catalase, exploring the role of Tyr42 in stabilizing the dimanganese cluster and implications for the involvement of the bridging ligands in the catalytic mechanism.

Molecular biology and biochemical preparations

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

Genomic DNA was isolated from Lactobacillus plantarum (ATCC 14431) and Lactobacillus brevis (ATCC 8727) by standard methods. The pG+host5 plasmid [20,21] was kindly provided by Emmanuelle Maguin, INRA, France. The Mn catalase structural gene with its native promoter was PCR amplified from L. plantarum genomic DNA (using the primers 5′-GCGAGGATCCAACCGACTATTGACTGGTAAAAAAGCAGTTACCCCTAACCAG-3′ and 5′-GAGCGAATTCCCACCTCCAATTTGAAATAGCCACCGCC-3′), BamHI/EcoRI digested and ligated into similarly digested pG+host5 plasmid. The pG+host5LPC product was recovered from an E. coli transformant and transformed into a kat strain of L. plantarum (NCDO 1193) by electroporation. Electrocompetent L. plantarum cells were prepared as previously described [22]. The protein expression level was subsequently improved by construction of a second expression vector based on the pMG36e plasmid [23], using the L. brevis surface layer protein promoter (PslpA) [23,24] in place of the native katM promoter. A 267 nucleotide fragment of the 5′ UTR for L. brevis slpA including the dual slpA promoter was PCR amplified from genomic DNA using the primers 5′-CGTGAATTCGATTACAAAGGCTTTAAGCAGGTTAGTGACGTTTTAG-3′ and 5′-CGTTCTAGACATATGCTTTCTTCCTCCAAACATAAAATATGTAATTTATCAAGCAAG-3′, the latter providing a convenient NdeI/XbaI linker site for ligating inserts for expression, precisely aligning the start codon under the ribosomal binding site in the slpA 5′ UTR. The PCR product was digested (EcoRI/XbaI) and ligated to similarly digested pMG36e to form pMG36ePslpA. A single NdeI site occurring in the transcriptional repressor gene (repA) coding region in the vector arms was eliminated by silent mutagenesis using the QuikChange site directed mutagenesis procedure (Stratagene, La Jolla) using the primer 5′-GTTGAGATACTTGATTATATCAAAGGTTCTTATGAATATTTGACTCATGAATC-3′ and its complement. The LPC structural gene (katM) was PCR amplified from pG+host5LPC using the primers 5′-CCGCATATGTTCAAACATACAAGAAAACTGCAATACAACGCAAAACC-3′ and 5′-CGCTCTAGATTATTAACCTTGGTGGTTGTGTAATCTAGGATCACCCG-3′, the latter including a double terminator sequence (TAATAAT). The instability of the pMG36ePslpA plasmid that has been observed in E. coli[25] was eliminated by reversing the orientation of the expression cassette in the plasmid (details to be reported elsewhere). The latter plasmid (pVMG36PslpA-LPC) was used for all protein expression described in this study. The Y42F mutation was introduced in this plasmid by QuikChange site directed mutagenesis using the mutagenic primer 5′-CCACTGGGATGATGTCTTTTCTCTCACAAGGTTGGGCG-3′ and its complement.

Protein expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

L. plantarum was grown at 37 °C in a New Brunswick Scientific BioFlo 3000 Bioreactor equipped with a 14-L fermentation vessel. The culture medium was 10 L of MRS medium supplemented with 2% glucose, 1%l-(–)-malic acid, 5 mm MnSO4, 0.5 mm CaSO4, 1 g Tween 80, 30 mg thiamine hydrochloride, and 50 mg erythromycin. The pH of the culture was regulated (pH 5.5) by addition of 15% NH4OH and sparged with air to maintain 20% dissolved oxygen level. Cells were collected by centrifugation at late log phase (D600 > 10) yielding approximately 400 g of slimy wet cells. Manganese catalase was isolated as previously described [7,8,26]. The Y42F mutant protein was detected in chromatographic fractions and its purity estimated during purification by electrophoresis (SDS/PAGE). The Mn content of the purified protein was determined by atomic absorption analysis using a Varian Instruments SpectrAA 20B graphite furnace atomic absorption spectrometer. Catalase activity was measured by the optically detected decomposition of hydrogen peroxide [8] and the Km for H2O2 was determined using a Clark oxygen electrode [7]. The homogeneous reduced (2,2) state of manganese catalase was prepared by treating the protein with a five-fold excess of (NH2OH)2·H2SO4 for a half hour followed by desalting on a gel filtration column. To convert the enzyme to the homogeneous (3,3) state, a solution of the reduced protein (10 mg·mL−1 in 50 mm potassium phosphate buffer pH 7, total volume <5 mL) was sealed in a 125-mL serum bottle under pure oxygen at 8 °C for one week. The oxidation half-time at room temperature was estimated by following the optical absorption of the enzyme in a sealed cuvette under O2 purge following reduction and anaerobic desalting. The reductive titration of manganese (3,3) catalase was performed as previously described [27] using (NH2OH)2·H2SO4 as titrant.

Spectroscopic measurements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

Optical absorption spectra were recorded on a Varian Instruments Cary 5E UV-vis-NIR absorption spectrometer. CD and MCD spectra were recorded using an AVIV Associates model 40DS UV-vis-NIR dichrometer as previously described [28]. An Oxford Instruments SM4-6T magnetocryostat provided the magnetic field and temperature control during the MCD experiments. EPR measurements were performed using a Bruker E500 X-Band EPR spectrometer equipped with a SuperX high stability bridge and a Bruker ER4116 Dm bimodal microwave resonator and an Air Products helium flow cryostat.

X-ray crystallography

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

Crystallization. Crystals of Y42F LPC were grown from 14% PEG 8000 in 0.1 m Taps buffer pH 8.7 by the vapor diffusion technique at 18 °C. Crystals used for structural studies were 0.4 × 0.2 × 0.15 mm belonging to space group P21 with unit cell dimensions a = 73.49, b = 95.27, c = 105.00, β = 106.55°.

Data collection and processing. Data was collected on a magenta crystal of Y42F manganese catalase flash-frozen at 100 K nitrogen stream after soaking for 1 min in a cryoprotectant solution [12% PEG 8000, 0.1 m Mes pH 5.5 and 30% (v/v) 2-methyl-2,4-pentandiol (MPD)]. Data sets at 1.75 Å (low resolution) and at 1.33 Å (high resolution) were collected at 100 K on station PX 14.2 at the Daresbury Laboratory (UK) equipped with an ADSC Quantum4 CCD detector on the same crystal and the two datasets were indexed and integrated using the program denzo[29] and scaled separately by scalepack[29]. Scaled data were merged by scalepack to construct a complete high-resolution dataset. Intensities were transformed into amplitudes using the program truncate of CCP4 suit [30]. The details of data collection statistics are presented in Table 1. Microscopic inspection of the crystal after data collection showed no difference in color between exposed and unexposed parts of the crystal, confirming the absence of significant photoreduction of Mn(III) centers during the X ray measurements.

Table 1. Summary of crystallographic statistics for Y42F Lactobacillus plantarum manganese catalase.
Data sets Y42F LPCLow resolutionHigh resolutionMerging of two data sets
  1. a Numbers in parentheses indicate values for the highest resolution shell. b Rsym = Σ[|II − 〈Ii〉|]/Σ[〈Ii〉]; c Rcryst = Σ||Fobs| −|Fcalc||/Σ|Fobs|; d Rfree[35] is the same as Rcryst [36] for a random subset not included in the refinement of 5% of total reflections.

Space groupP21P21 
Unit cell parameters Å
a73.4973.49 
b95.2795.27 
c105.00105.00 
β106.55106.55 
Temperature (K)100100 
mosaicity (degrees) 1.05 
B-value from Wilson plot (Å2)  14.28
Resolution limits (Å)50.0–1.7523.00–1.33 
Last shell limits (Å)1.79–1.751.35–1.33 
Completeness (%) a86.3 (71.7)86.7 (80.7) 
Reflections305406588079395381
Unique reflections120714274725282601
Redundancy2.532.14 
I/s14.2 (2.8)14.9 (1.7) 
Rsym (%) b4.3 (22.0)4.1 (34.0)Rmerge 3.9
Final refinement statisticsY42F LPC  
Resolution limits (Å)48.0–1.33  
Refined number of reflections268292  
Number of reflections for Rfree14165  
Overall Rcryst (%) c11.5  
Overall Rfree (%) d14.5  
Number protein residues1596  
RMS bonds (Å)0.014  
RMS angles (degrees)1.522  
Average B-value (Å2)16.28  
Total nonhydrogen refined atoms13957  
Protein Atoms12450  
Solvent Atoms1507  
Mn+3 Ions12  
Ca+2 Ions6  

Structure determination . The structure of LPC Y42F mutant was solved using coordinates of the WT LPC structure [PDB ID 1JKV] as the initial model for program refmac[31] in Rigid Body mode, followed by positional and temperature factor refinement. Atomic positions and individual temperature factors were refined simultaneously using refmac with the maximum-likelihood target function for amplitudes and the exponential bulk-solvent and anisotropic overall temperature factor correction. The six monomers of the Y42F homohexamer were refined independently. Water molecules were improved using the program ARP [32]. Mes buffer molecules and multiple side chain conformations were modeled in this stage during inspection of waters. Occupancy of multiple side chain conformation was validated manually by investigating the difference density of FoFc maps. Rebuilding of model was carried out with the program O [33]. Hydrogen atoms in standard positions were included in the final stages of refinement followed by anisotropic B-factor refinement by refmac[34]. The current R factor of this model is 11.5% (Rfree = 14.5%). An example of the final 2Fobs– Fcal electron density map at 1.33 Å resolution is shown in Fig. 3. The model of Y42F LPC has 91.8% of the residues in the most favored regions of the Ramachandran plot and 8.2% in the additionally allowed regions. Some characteristics of the refined models are presented in Table 1.

image

Figure 3. Stereoview of fragments of the electron-density maps including the dimanganese active site. The omit map was calculated with the complete Y42F LPC model not including the alternative conformation for the Glu35 and Glu148 side chains. The electron-density of 2FoFc map is contoured at 1.3 σ (light blue line) and 10.0 σ (dark blue line). The difference density of FoFc map is rendered as red lines at 4.0 σ. This figure was generated with the program bobscript[37,38].

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Biochemical characterization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

The yield of purified manganese catalase was approximately 120 mg from 10 L of culture for homologous expression of recombinant enzyme from L. plantarum containing the pVMG36PslpA-LPC expression plasmid, five times higher than observed for cells containing the pG+host5LPC vector where the LPC gene expression is under the control of the native promoter. Y42F LPC exhibits a slightly lower metal content (1.4 Mn/active site) than is found in the recombinant WT protein (1.7 Mn/active site). Manganese clusters in the as-isolated recombinant WT and Y42F LPC are predominantly in the oxidized (3,3) state, based on optical absorption measurements (vide infra). EPR spectra of the as-isolated Y42F LPC (data not shown) indicate that a significant fraction of sites (although less than 10% of the total) are present in the superoxidized, mixed valent (3,4) state. As previously found for WT manganese catalase [27], the (3,3) and (3,4) clusters in the Y42F LPC can be reduced by hydroxylamine to a homogeneous (2,2) state. The reduced clusters may then be cleanly reoxidized by O2 to a homogeneous (3,3) state at pH 7 or higher (to pH ≈ 9.5). At 8 °C, the oxidation approaches completion after 1 week at pH 7 for both WT and Y42F LPC (data not shown). The half-time (t½) for the re-oxidation reaction was estimated as 24 h for WT and 23 h for Y42F LPC at 8 °C, and 8 h for WT enzyme and 14 h for Y42F LPC at room temperature.

Y42F LPC retains a few per cent of the catalase activity exhibited by the WT enzyme (Table 2), with catalytic activity increasing at elevated pH. This small residual activity of the mutant enzyme has been consistently observed for all preparations, and is not observed in the extracts of cells not expressing Y42F LPC. The pH sensitivity of the catalase activity of Y42F LPC contrasts with the behavior of the WT enzyme whose activity varies only slightly over the pH range 5–10. At neutral pH the Km value for Y42F LPC was significantly higher than that of WT enzyme (1.4 m vs. 220 mm). At pH 10, on the other hand, the Km value of Y42F LPC was dramatically lowered (to 220 mm), becoming comparable with that of the WT enzyme which was relatively unaffected by raising the pH.

Table 2. pH Dependence of catalytic activity for WT and Y42F manganese catalase.
Assay pHSpecific activity (U·mg−1)
Recombinant WT LPCY42F LPC
  1. a 50 mm sodium dimethylsuccinate, 0.1 mm EDTA; b 50 mm potassium phosphate, 0.1 mm EDTA; c50 mm Taps/KOH, 0.1 mm EDTA; d50 mm Caps/KOH, 0.1 mm EDTA.

5.0a7688 ± 10757 ± 2
6.0b8416 ± 329178 ± 6
7.0b8859 ± 536252 ± 3
8.0b8258 ± 413452 ± 5
9.0c7957 ± 376584 ± 10
10.0d8914 ± 528898 ± 24

X-ray crystal structure of Y42F LPC

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

The structure of Y42F LPC is nearly identical to that of the WT LPC, with a RMS deviation between the aligned polypeptide chains of only 0.26 Å over all backbone atoms. Even in the region of the mutation, the basic fold of the WT protein is preserved, but the deletion of the hydroxyl group in the aromatic side chain on substitution of phenylalanine for tyrosine in Y42F LPC perturbs the hydrogen bonding interactions in the outer sphere environment of the active site manganese cluster, locally altering the protein structure (Fig. 4). Loss of hydrogen bonding interactions with its donor/acceptor partners (Arg147/Glu148) has only a small affect on the conformation of residue-42 itself. Phe42 in Y42F LPC is only slightly shifted relative to the framework of the polypeptide backbone from the position occupied by the aromatic sidechain of Tyr42 in WT LPC, apparently being held in place by buttressing contacts with a cluster of buried residues. Deletion of the phenolic hydroxyl in the Y42F mutant protein leaves a void between the aromatic side chain of Phe42 and the Glu148 carboxylate that is not occupied by solvent. Based on the intense color of the crystalline protein and the relatively short metal-ligand bond distances observed in the structure, the dimanganese sites are assigned the oxidized (3,3) state in the crystal. All Mn sites in the crystal appear to be fully occupied. The mean temperature factor of the Mn ions is 19.5 Å2 (close to the mean temperature factor for the protein structure, 18.0 Å2), and no negative difference electron density is detected in the FobsFcalc map of the cluster core assuming full occupancy of the metal binding sites. As the metal content of the as-isolated Y42F LPC is substoichiometric (70–88% Mn depending on method of analysis), this suggests that crystallization selects fully metallated hexamers in the lattice. The average inner sphere bond distances for the two manganese subsites in the cluster (Table 3) are nearly identical to those found for the native LPC dimaganese complex, confirming that the fully oxidized Mn(III)Mn(III) core is represented in the crystal structure. The slight decrease in average metal-ligand distance for subsite 2 is consistent with the lower coordination number (CN) for that metal center in Y42F LPC (CN = 5 for Mn2 in Y42F LPC, compared to CN = 6 in WT LPC). The perturbation of the cluster core results in a disruption of the Mn2-W2 bond and leads to smaller changes in several other ligand interactions, which combine to produce the overall shift of Mn2 towards the Glu148 carboxylate headgroup.

image

Figure 4. Stereoview of aligned WT and Y42F LPC structures showing the affect of Y42F mutagenesis on the extended outer sphere environment of the dimanganese cluster. The region shown represents residues within a 10-Å of the CD carbon atom of Glu66 following alignment (superposition) of all backbone atoms of the enzyme (with an RMS deviation of 0.14 Å). Color coding is used to distinguish the individual protein chains [WT LPC (PDB ID 1JKU), red; Y42F LPC (PDB ID 1O9I), green] and heteroatoms (WT LPC: Mn ions, red; solvent molecules, violet; Y42F LPC: Mn ions, blue; solvent molecules, yellow). Alternative conformations of the Glu35 and Glu148 side chains are rendered in blue.

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Table 3. Inner sphere bond distances for WT and Y42F LPC Mn centers.
Bond vectorMetal-ligand bond distance (Å) a
WT LPC bY42F LPC c
  • a

    Underlined values represent distances with >5% change between WT and Y42F LPC structures;

  • b

    b PDB ID 1JKU;

  • c

    PDB ID 1O9I.

Subsite 1
 Mn1-W11.912.09
 Mn1-W22.102.07
 Mn1-W32.062.09
 Mn1-Glu35OE21.881.87
 Mn1-Glu66OE12.132.12
 Mn1-His69ND22.202.18
 Avg2.052.07
Subsite 2
 Mn2-W12.002.11
 Mn2-Glu66OE22.082.01
 Mn2-Glu148OE12.442.30
 Mn2-Glu148OE22.362.13
 Mn2-His181ND22.142.10
 Avg2.202.13

Arg147 is released from its interactions with residue-42 in Y42F LPC, and its guanidinium headgroup undergoes a shift (0.28 Å) away from the cluster, perturbing its hydrogen bonding interactions with the solvent bridge (W2) and one of the metal ligands (Glu35). The latter side chain is slightly displaced away from the cluster (by 0.33 Å) along the cluster axis. The nonligand carboxylate Glu178 headgroup also shifts away from the cluster (by 0.63 Å) through a small (11°) twist in the extended sidechain. In spite of these significant perturbations of the active site environment, both ligating histidine residues (His69 and His181) as well as Glu66, which contributes the bridging carboxylate for metal binding, are nearly invariant between the WT and Y42F LPC. More surprisingly, the Glu148 ligand, which loses one of its hydrogen bonding interaction in the mutant, remains rigidly fixed relative to the polypeptide framework in the major conformation present in the crystal. (An alternate conformation of the Glu35/Glu148 pair is also detected in the crystal structure.)

Although the position of Glu148 in the major conformer is not significantly affected by Y42F mutagenesis, its altered reactivity is reflected in dramatic changes in the cluster core (Fig. 5). Only one of the solvent molecules (W1) associated with the dimanganese core remains bridging in the Y42F LPC cluster. The solvent (W2), which formed a second bridge in the WT structure, is terminally bound to Mn1 in the mutant complex to form an octahedral coordination polyhedron for this subsite. The position of W2 in Y42F LPC is roughly the same as found for the corresponding atom in WT LPC, but the second manganese ion (Mn2) is displaced towards Glu148 ligand in the Y42F LPC complex, allowing it to be more symmetrically chelated by the carboxylate headgroup. In the WT enzyme, the OE1 oxygen of Glu148 is closest to Mn2, while in Y42F LPC the OE2 oxygen is closest, resulting in distorted trigonal bipyramidal coordination for the Mn2 subsite, the OE2-Mn2-W1 direction defining the trigonal axis. This change in coordination chemistry for Glu148 suggests that an increase in ligand basicity resulting from loss of the tyrosine hydrogen bond underlies the reorganization of the cluster core. The detailed metric features of the dimanganese core for Y42F LPC are indicated in Scheme 1.

image

Figure 5. The active site of Y42F Lactobacillus plantarum manganese catalase. Manganese ions (Mn1, Mn2) and coordinated solvent molecules (W1, W2, W3) are labeled and hydrogen bonds inferred from the structure are shown. Conformations A (top) and B (bottom) are shown (based on PDB ID 1O9I). The structures were rendered using ortep-3 [13].

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image

Figure Scheme 1.. Metric parameters of the Y42F LPC dimanganese core.

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In addition to the active site conformation (A) which closely resembles the organization of the WT LPC active site, the ligating glutamate residues Glu35 and Glu148 are found to adopt a second conformation (B) in the crystal (Fig. 5), based on a distinct pattern of hydrogen bonds. In conformation B, both carboxylate head groups are rotated approximately 90°[with OW3-Mn1-OE2-OE135 dihedral changing from 89.12° (A) to −15.3° (B) and N181-Mn2-OE2-OE1148 dihedral changing from −96.3° (A) to 167.4° (B)], allowing the OE2 oxygen of Glu148 to serve as a hydrogen bond acceptor to both the NE and NH2 nitrogens of Arg147, substituting for the missing Y42 phenolic oxygen. This structural variation is reminiscent of the carboxylate shift isomerism observed in the diiron carboxylate family of proteins [39–42]. The change in binding mode of Glu148 from bidentate chelating to monodentate syn coordination lowers the coordination number of the Mn2 subsite to four, with distorted tetrahedral geometry. Metal-ligand bond distances for Glu35 and Glu148 OE2 carboxylate donor atoms are slightly longer in conformation B [Mn1-O35(B) 2.23 Å, Mn2-O148 (B) 2.22 Å]. Reorientation of Glu35 allows a hydrogen bond to be formed with the terminally bound solvent (W3). Conformation A is expected to be stabilized relative to conformation B by two additional hydrogen bonds in the outer sphere of the cluster, which may account for its slight predominance in the mixture (a 60 : 40 ratio based on occupancy estimates in the crystallographic analysis). The uniform behavior of Y42F LPC in spectroscopic measurements and active site titration experiments (see below) suggests that it is not possible to distinguish these two conformers in solution samples.

Spectroscopic characterization – optical absorption spectroscopy

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

The optical absorption spectrum of Y42F LPC in the (3,3) state (Fig. 6, spectrum 1) exhibits an absorption maximum near 480 nm with an extinction coefficient of 1100 m−1·cm−1 at pH 6 (ε480 = 700 m−1·cm−1 per Mn) and weaker absorption maximum at 750 nm (Table 4). The stronger absorption band includes a shoulder near 560 nm and fine structure features between 500 and 530 nm arising from spin-forbidden electronic transitions. A quantitative stoichiometric redox titration of the O2 re-oxidized Y42F mutant protein with (NH2OH)2·H2SO4 demonstrates a uniform reduction process yielding an estimate of 0.88 ± 0.03 sites in the (3,3) state per LPC monomer (Fig. 6, inset). The optical extinction coefficient for the visible absorption bands decrease with increasing pH, and at pH 10 the spectrum (Fig. 6, spectrum 2) resembles that of the WT enzyme but with higher absorptivity (ε480 = 350 m−1·cm−1 compared to 200 m−1cm−1 for WT LPC) (Fig. 6, spectrum 3).

image

Figure 6. Optical absorption spectra for Lactobacillus plantarum manganese catalase (3,3) complexes. (1) Native Y42F LPC (0.25 mm active sites) in 50 mm potassium phosphate pH 6; (2) Y42F LPC (0.25 mm active sites) in 50 mm Caps/KOH pH 10; (3) Native WT LPC manganese catalase (1 mm active sites) in 50 mm Mops pH 7. (Insert) Stoichiometric redox titration of Y42F (3,3) LPC (0.23 mm active sites in 50 mm potassium phosphate pH 7) under argon was titrated with aliquot addition of an anaerobic aqueous solution of (NH2OH)2·H2SO4.

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Table 4. Spectroscopic properties of Y42F LPC (3,3) complexes.
Y42F LPCABSCDMCD
λ (nm)ε (m−1·cm−1)λ (nm)Δε (m−1·cm−1)λ (nm)Δε (m−1·cm−1)
pH 6
 4801100490−8.3350−9.7
 750100560−9.8395−4.7
   750+1.7460+47.8
     505−3.5
     515+8.5
     560−32.4
     735+2.1
pH 10
 475490450+2.8
   500−2.5  
   550−3.4  

CD and MCD Spectra

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

The CD spectrum (data not shown) of Y42F LPC ligand-free enzyme in the (3,3) state shows a doublet pattern of negatively signed intensity at 490 and 560 nm as well as weaker positively signed ellipticity near 750 nm (Table 4). The high pH complex exhibits dramatically lower CD intensity near 500 nm and loss of the doublet splitting pattern.

At low temperatures (5 K) and high magnetic fields (4 T) (3,3) Y42F LPC complex exhibits unusually strong MCD spectra, with well-resolved features at 460 and 560 nm and a pair of weak, sharp features at 505 and 515 nm (Fig. 7). A saturation profile for the 460 nm MCD feature (Fig. 7, insert) reveals strongly nested magnetization curves for temperatures between 2 and 8 K. The high pH form of the (3,3) Y42F LPC complex shows virtually no paramagnetic MCD over the entire UV-vis-NIR absorption range (Fig. 8), although a pattern of weak features closely resembling the oxidized (3,3) complex of WT LPC [27] in both transition energies and intensities remains: ΔεL-R = −2.4 m−1·cm−1·T−1 (350 nm); +3.1 m−1·cm−1·T−1 (430 nm); −1.5 m−1·cm−1·T−1 (495 nm); −1.6 m−1·cm−1·T−1 (550 nm).

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Figure 7. Magnetic circular dichroism data for Y42F Lactobacillus plantarum manganese catalase. (Left) MCD data for native Y42F LPC (top) Variable-magnetic field MCD spectra for (3,3) Y42F LPC (1.2 mm active sites) in 50 mm potassium phosphate pH 6, 50% glycerol glass, T = 5 K. (···) 4T LT-MCD spectrum for WT LPC (3,3) complex included for comparison. (Insert) Saturation-magnetization profiles for low temperature (2–8 K) variable magnetic field (0–5 T) MCD. A splitting diagram for non-Kramers ground state with large fine structure splitting (δ) is shown.

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image

Figure 8. Magnetic circular dichroism data for Y42F Lactobacillus plantarum manganese catalase (3,3) high pH form. Variable-magnetic field MCD spectra for native Y42F LPC (1 mm active sites) in 30 mm Caps/KOH pH 10, 50% glycerol glass, T = 5 K. Dashed line, 10 ×expansion (right hand scale).

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EPR spectra of the (3,3) complex

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

Low temperature (10 K) EPR spectra for oxidized (3,3) Y42F LPC (Fig. 9) exhibit an intense, extremely low-field resonance (geff = 20) extending to the zero-field limit of the spectrum. This feature appears in both perpendicular (Fig. 9, solid line) and in parallel (Fig. 9, dashed line) EPR polarization. In parallel polarization, extended hyperfine modulation of the resonance is partly resolved, with an average effective hyperfine splitting aMn = 37 G. In addition to this dominant resonance feature, a number of other spectral components centered near geff = 6 (sextet), geff = 4.3 (complex multiplet) and g = 2 (sextet) are also observed in perpendicular polarization, consistent with the presence of several minority species in the enzyme sample. The weakest component, near geff = 6, is observed in both parallel and perpendicular polarization, and the modulation pattern is consistent with an average hyperfine coupling aMn = 85 G. A complex pattern of hyperfine features centered near geff = 4.3 in perpendicular polarization appears to be comprised of two overlapping subspectra of similar intensity (Fig. 9, A and A′), each exhibiting a sextet multiplet splitting (aMn = 93 G). An EPR signal near g = 2 present in the spectrum of Y42F LPC also clearly exhibits sextet multiplet splittings, and neither the geff = 4.3 nor g = 2 spectra are observed in parallel polarization. Thus, polarization experiments allow the even-electron systems [mononuclear Mn(III), and homovalent (2,2) and (3,3) complexes] to be distinguished from mononuclear Mn(II) sites in the protein.

image

Figure 9. EPR polarization spectra for Lactobacillus plantarum manganese catalase (3,3) complex. Native Y42F LPC (3,3) (2.5 mm active sites) in 50 mm potassium phosphate pH 6. A, A′ denote sextet components. Solid line, perpendicular polarization; dashed line, parallel polarization. Instrumental parameters: microwave frequency, 9.68 GHz (perpendicular) or 9.41 GHz (parallel); microwave power, 10 mW; modulation amplitude, 5 G; modulation frequency, 100 kHz; temperature 8.5 K.

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High pH converts the (3,3) Y42F LPC to a distinct complex, with relatively weak low-field EPR resonances in both parallel and perpendicular polarizations (Fig. 10). In parallel polarization a distinct hyperfine pattern is resolved in the low-field resonance, with an average aMn = 32 G. The A + A′ multiplet persists in this sample, and the region near g = 2 appears to be a superposition of the sextet feature observed at neutral pH and the multiline spectrum from a minor mixed-valent (3,4) component.

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Figure 10. EPR polarization spectra for Y42F Lactobacillus plantarum manganese catalase (3,3) hydroxide complex. Native Y42F LPC (3,3) (2.3 mm active sites) in 50 mm Caps/KOH pH 10. Solid line, perpendicular polarization; dashed line, parallel polarization. Instrumental parameters: microwave frequency, 9.68 GHz (perpendicular) or 9.41 GHz (parallel); microwave power, 10 mW; modulation amplitude, 5 G; modulation frequency, 100 kHz; temperature 8.5 K.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

Conserved tyrosine residues are present in the outer sphere of metal centers in a wide range of metalloproteins, including ribonucleotide reductase (RNR), the oxygen evolving complex (OEC) of photosystem II, prostaglandin H synthase, catalase, ferritin, and superoxide dismutase. In some cases (e.g. the Tyr122 in RNR [43]; YZ, assigned to Tyr161 of the D1 protein in the OEC [44]; Tyr385 of ovine prostaglandin H synthase-1 [45]) the tyrosine residues are redox active, forming tyrosyl phenoxyl free radicals that serve as hydrogen atom abstraction sites storing oxidation equivalents in the protein. The function of conserved tyrosines in other metalloproteins is less clear. In ferritin [46], Tyr34, a residue adjacent to the binuclear iron ferroxidase active site, has been proposed to serve an electron transfer role in analogy to RNR where Tyr122 appears in a similar context. In iron and manganese-cofactored superoxide dismutases, the conserved tyrosine residue occupies a position at the base of the substrate access funnel [47,48], and may contribute to substrate interactions, facilitate proton transfer steps, or serve as a safety valve preventing permanent oxidative damage to the protein through reversible formation of a phenoxyl species. Conservative substitution of phenylalanine for tyrosine in ferritin [46] and manganese SOD [49,50] has negligible effects on turnover under physiological conditions, suggesting that evolution may have selected the conserved tyrosines for function under extreme or unusual conditions. Sequence correlations within the manganese catalase family of enzymes have revealed a highly conserved tyrosine (Tyr42 in LPC sequence numbering) preceding the first EXXH metal binding motif by approximately 25 residues. X-ray crystallography of both Thermus thermophilus[10] and Lactobacillus plantarum[11] manganese catalases has shown that this tyrosine is intimately associated with the dimanganese core, lying in the outer sphere of the metal cluster and forming a hydrogen bond with one of the manganese ligands. The significance of this interaction is unknown, and we have prepared Y42F LPC using site directed mutagenesis and expressed the mutant protein in a homologous expression system in order to address the function of Tyr42 and investigate the role of the outer sphere tyrosine in manganese catalase structure and reactivity.

Replacement of Tyr42 by phenylalanine does not interfere with the assembly of the dimanganese center in LPC, and recombinant Y42F LPC has only slightly less metal content than recombinant WT LPC. X-ray crystallography reveals that mutagenesis perturbs the structure of the binuclear active site, breaking one of the solvent bridges (W2). This loss of a bridge from the cluster core may be traced, in turn, to a disruption of the hydrogen bonding web around the active site. Deletion of the Tyr42 phenolic hydroxyl in Y42F LPC removes the hydrogen bonding contact with the Glu148 ligand to Mn2, altering the basicity of the carboxylate group and significantly affecting its coordination behavior (Fig. 5). This contributes to the expansion of the intermanganese distance from 3.03 Å in the (3,3) state of the WT enzyme to 3.33 Å in the corresponding state of Y42F LPC, as Mn2 is drawn toward the chelating Glu148 headgroup (Scheme 1). The W2 bridge is further destabilized in the mutant by the loss of the Arg147-Tyr42 hydrogen bond that rigidly anchors Arg147 in WT LPC, allowing W2 to dissociate as the Mn-O-Mn angle is opened from 101.6° to 116.1°, resulting in extremely asymmetric coordination of the W2 solvent in an ‘open’ form of the cluster. An open, single-atom-bridged dimanganese cluster has previously been proposed to be the catalytically active form of LPC and the major form of LPC in solution [51], although the closed form of the cluster (containing two atom bridges) has been reported for in the WT LPC crystal structure [11] consistent with detailed spectroscopic characterization of the WT LPC in solution [27]. These effects of Y42F mutagenesis demonstrate a clear role for Tyr42 in stabilizing the native bridged manganese cluster structure in LPC.

Alteration of the dimanganese core structure in Y42F LPC has dramatic effects on both the electronic structure of the cluster (as reflected in the perturbed spectra) and its catalytic reactivity. Although the open form of the cluster can be readily converted between the (2,2) and (3,3) oxidation states that form the basis for the catalytic reaction cycle in the WT enzyme [52,53], Y42F LPC supports <1% of the rate of WT peroxide dismutation at low pH (Table 2), indicating that Tyr42 is required for full catalytic activity. However, the rates for reoxidation of the WT and mutant clusters by dioxygen, which likely occurs through the reverse of the reductive turnover half-reaction, are similar for the two proteins, although the reoxidation rates of both proteins are much slower than the catalytic rates (data not shown). One of the most dramatic effects of Y42F mutagenesis is on the Km for substrate, H2O2, which is increased at neutral pH from 220 mm for WT enzyme to ≈ 1.4 m in the mutant. At pH 10, the Km value for Y42F LPC approaches the value for the WT enzyme, while the Km value for WT enzyme decreases only slightly (from 220 mm at pH 7 to 160 mm at pH 10). The specific activity of the mutant protein also exhibits significant pH dependence (Table 2), in contrast to the WT enzyme. These differences in pH sensitivity reflect differences in coordination chemistry for the open and closed forms of the dimanganese cluster.

Loss of one of the solvent bridges fundamentally alters the properties of the manganese ions and their electronic interactions within the cluster, as demonstrated by the unique spectroscopic characteristics of the Y42F active site complexes. The oxidized (3,3) Y42F LPC exhibits unusually strong absorptivity in the ligand field spectra for the Mn(III) centers at low pH (Fig. 6), at least fourfold higher than found for the WT enzyme, consistent with a relaxation of the Laporte (orbital parity) selection rules for the d(r)d transitions under lower coordination number and lower symmetry of the individual metal environments in the mutant protein [28]. The CD spectrum of the ligand-free complex appears to resolve spectra for Mn(III) in each of the subsites of the cluster, giving rise to a distinctive ellipticity doublet. The spectroscopic nonequivalence of the two Mn centers in the Y42F LPC active site may be understood in terms of the crystallographically determined metal environments. In the Y42F LPC cluster, both metal centers have noncentrosymmetric ligand distributions, with Mn2 (the five-coordinate subsite) being chelated by Glu148 while Glu35 is bound to Mn1 (the six-coordinate subsite) in a monodentate coordination mode, accounting for the differences in ligand field strengths reflected in the spectroscopic splittings. Both major and minor conformations of the two coordinating glutamate side chains lead to distinct coordination numbers for the two manganese ions in the cluster.

The ‘open’ cluster, containing a single atom bridge, also exhibits dramatically different ground state interactions between the two Mn(III) ions, reflecting the electronic coupling responsible for the two-electron reactivity of the cluster. In contrast to the diamagnetic (ST = 0) ground state characteristic of the (3,3) form of WT LPC (as well as TTC) [27], the exceptionally strong paramagnetic MCD associated with (3,3) Y42F LPC (Fig. 5) implies a ferromagnetically coupled ground state in which the ST = 4 multiplet is lowest in energy as has been found for other binuclear Mn(III) complexes with single atom bridges [54–56]. The saturation magnetization curves for the ligand-free Y42F complex (Fig. 7, insert) are strongly nested, typical of MCD for ground states exhibiting thermally accessible zero field splittings [57,58]. The ST = 4 state is a non-Kramers (integer spin) multiplet comprising nine MS sublevels (MS = 0, ± 1, ± 2, ± 3, ± 4) whose degeneracies may be completely removed in the absence of a magnetic field by ligand perturbations expressed in the zero field splittings (δ). The low temperature MCD behavior of Y42F LPC implies relatively large initial splittings are present within the electronic ground state multiplet, whether they are intradoublet zerofield splittings (δ) or interdoublet fine structure splittings, although complementary EPR measurements (see below) appear to support the latter.

We have previously shown that the ratio of saturation limit MCD intensity to absorptivity (the MCD saturation ratio, ΔεMCDABS) may be useful as a measure of the Faraday ratio C°/D° permitting a more quantitative interpretation of paramagnetic MCD spectra that is essential for interpretation of complex spectra from biological samples [27]. MCD and optical absorption spectra for the Y42F LPC complex (Fig. 7) yield ΔεMCDABS = 0.10, similar to the values previously found for high spin Mn(III) complexes of Mn superoxide dismutase [28] and the fluoride derivative of WT LPC [27]. On the other hand, the value found for the high pH form of Y42F LPC (Fig. 8) is relatively small (approximately 0.01), consistent with ≤ 10% of the open cluster remaining at pH 10. Similar evaluation of the small paramagnetic MCD signal that is present in WT LPC (3,3) samples allows us to estimate that less than 3% of the sites occur in the ‘open’ form in the WT enzyme. This clearly illustrates the importance of quantitative characterization of MCD samples, as this minor impurity species was interpreted as the sole component in earlier studies of the oxidized (3,3) state of LPC [51].

EPR spectra of the oxidized Y42F LPC complement the MCD results and support assignment of a ferromagnetically coupled ground state for the (3,3) complex in the mutant protein (Fig. 9). Conventional low temperature EPR spectra recorded in perpendicular polarization (H1 ⊥ H0) are dominated by a strong resonance extending to the zero field limit, which is also present in the parallel polarized spectra (H1 || H0). Under selection rules for microwave absorption in parallel polarization (ΔMS = 0) [59], transitions between pure angular momentum states (different MS values) are not allowed, and therefore spectral components associated with minor impurities containing half-integer spin (Kramers) metal ions (e.g. mononuclear Mn(II), 3d5, S = 5/2) are strictly forbidden. Transitions between mixed MS states (linear combinations of the pure angular momentum functions that arise through orbital mixing) are, however, allowed in parallel polarization if the same MS function appears in both initial and final states, with the resonance condition (Eqn 1).

  • image(1)

defining the effective g-value at which EPR transitions are observed. As indicated in Eqn (1), Zeeman splitting of zero-field split sublevels is a second-order process depending on the magnitude of the initial splitting δ whose sensitivity to molecular environment leads to variation between individual molecules in a sample, resulting in relatively broad resonances. The behavior of the low-field resonance in (3,3) Y42F LPC is consistent with a transition between sublevels of a non-Kramers ground state with δ≈ 0.3 cm−1 (the X-Band microwave photon energy). This feature has a very strong temperature dependence, decreasing in intensity more rapidly than predicted by the Curie Law, implying that other nearby states are thermally accessible at low temperature (10–25 K).

The extended hyperfine pattern associated with this resonance has a major splitting (37 G) approximately half the value expected for a Mn(III) center (aMn = 80–90 G), consistent with assignment to the coupled binuclear (3,3) active site. In the exchange-coupled complex, the magnitude of the hyperfine splitting is reduced by projection of the individual ion spins (S1 and S2) on the total cluster spin (ST) [60]:

  • image(2)

As a consequence, the magnitude of the observed Mn hyperfine splitting for each ion is approximately half the single-ion intrinsic value within every multiplet state of the coupled complex. The observed splitting implies an intrinsic aMn = 74 G which is low for Mn(III), and may reflect relatively strong covalent delocalization associated with the oxo bridge, as well as the other ligands binding the cluster. In contrast, the EPR spectrum of the WT LPC (3,3) complex lacks any significant resonance features in either perpendicular or parallel polarization, other than a minor mixed valent impurity signal [27].

In combination with the MCD results discussed above, the EPR data indicate a paramagnetic ground state for the exchange-coupled complex that requires ferromagnetic exchange coupling of the two high spin Mn(III) metal ions in Y42F LPC. In contrast, within the doubly bridged, WT dimanganese core, the metal ions experience strong antiferromagnetic interactions, supported by the pair of atom bridges. This difference emphasizes the importance of the ground state magnetism as an indicator of the interactions within the cluster core. The change in the sign of the exchange coupling in the closed/open transformation may be traced to the loss of one of the atom bridges and its effect on the electronic coupling between the two metal ions. The orbital quantization axes of the two metal centers in the cluster are defined by the Mn(III)-oxo overlaps, representing the strongest ligand interactions in the complex. These strong σ antibonding overlaps with the Mn(III) metal centers destabilizes the dz2 valence orbitals on each ion, leaving them unoccupied in the d4 metal complex. The linear combination of the (empty) dz2 metal orbitals and the pσ on the oxygen bridge comprises an MO that represents the most important (σ) pathway for electronic coupling between the two metal centers, and the absence of unpaired electrons in the metal contributions to this MO eliminates the major antiferromagnetic contribution to exchange interactions in the complex. The opening of the Mn-O-Mn bridge angle in the cluster containing a single atom bridge further reduces the contributions from π pathways, producing a ferromagnetically coupled ground state for the µ-oxo bridged Mn(III) dimer. In the closed cluster, the second (hydroxo) atom bridge mediates strong antiferromagnetic coupling. Thus, spectroscopic and crystallographic results independently lead to the conclusion that one of the solvent bridges is broken in the oxidized Y42F LPC (3,3) complex.

In addition to the low-field EPR signal from the ferromagnetically coupled di-Mn(III) active site, other signals are also present in the sample (Fig. 9) that reflect a degree of heterogeneity among the sites. Resonances near geff = 4.3 and g = 2 appear exclusively in perpendicular polarization, and exhibit the sextet hyperfine splitting pattern expected for a mononuclear 55Mn metal ion (I = 5/2; 2I + 1 = 6). These components may be assigned to a minor fraction of half-occupied sites containing Mn(II) that is not readily oxidized by O2. The pattern of resonances near geff = 4.3 (A, A′) reflect the presence of Mn(II) in two similar but distinct metal environments, and may be associated with active sites in which either the Mn1 or Mn2 subsite is occupied. The large geff value suggests a relatively low-coordinate complex, similar to the five-coordinate Mn(II) sites in Mn superoxide dismutase. A third minor component, associated with resonances near geff = 6, also exhibits the sextet hyperfine splitting characteristic of mononuclear Mn site, but has significant intensity in the parallel polarized EPR spectrum, and therefore may be assigned to a small fraction of mononuclear Mn complexes containing an oxidized Mn(III) metal center.

The active site of Y42F LPC is much more sensitive to pH than that of the WT enzyme, and the optical absorption, MCD and EPR spectra indicate that at high pH the dimanganese complex in the mutant protein is converted to a form closely resembling that of the WT enzyme at neutral pH. This process likely involves coordination of hydroxide ion (HO) in the second bridge position, restoring the bis-bridged Mn(III) core characteristic of the WT active site (Scheme 2). Hydroxide binding would appear to be an exception to the observation that access to the LPC active site is restricted to neutral molecules [11,27], and a Grotthuss proton relay conduction mechanism [61] may be involved in this process. The high pH complex exhibits the relatively weak optical absorption of a six-coordinated Mn(III) center, and the absence of strong signals in either MCD (Fig. 8) or EPR (Fig. 10) spectra for the complex are consistent with antiferromagnetically coupled ground state as previously found for the WT enzyme. This structural transformation at high pH is mirrored in the pH dependence of the kinetic constants for Y42F LPC. We find distinct catalytic properties for the open and closed forms of the cluster: Km for H2O2 is lowered (eightfold) and the specific activity increased (threefold) at high pH, indicating that the closed form of the cluster is more effective in supporting peroxide dismutation. Note that WT LPC does not exhibit this type of pH sensitivity, so it seems unlikely that these properties are directly related to substrate ionization at high pH.

image

Figure Scheme 2.. Catalytic reaction cycle for manganese catalase turnover.

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The combined structural, spectroscopic and biochemical characterization of the dimanganese clusters in Y42F and WT LPC allow us to relate cluster structure to catalytic reactivity. The (3,3) state of the WT enzyme contains a catalytically active ‘closed’ cluster, associated with an antiferromagnetically coupled electronic groundstate mediated by a pair of solvent bridges. This structure is stabilized by the outer sphere hydrogen bonding network, including interactions with Tyr42. Disruption of the outer sphere hydrogen bonding network in Y42F LPC stabilizes a catalytically impaired ‘open’ form of the oxidized (3,3) cluster at neutral pH, reflected in the unique optical and magnetochemical properties of this site. The broken bridge (W2) appears to be rescued by exogenous hydroxide at high pH, regenerating the native, bis-atom bridged ‘closed’ form of the cluster core and slightly restoring catalytic function. In addition to this essential structural contribution of Tyr42, the proximity to the dimanganese cluster and the well-established redox properties of tyrosine residues in, e.g. ribonucleotide reductase, the outer sphere tyrosine in LPC may serve as a sacrificial redox site, a ‘safety valve’ capable of reversible one-electron oxidation protecting the inner sphere of the metal complex (particularly the histidine ligands) during relatively rare, high potential oxidative processes in the active site.

The observations described above for the Y42F LPC mutant provide the basis for new mechanistic insights, illustrated in Scheme 2. Each of the two solvent bridges in the WT LPC can serve as a single-base equivalent in proton-coupled electron transfer within the active site, with protonation being driven by perturbation of the bridge pKa by ligation or oxidation state changes at the metal centers. The pendant glutamate residue Glu178 that lies above the Mn2O2 core is positioned to form hydrogen bonds to ligands occupying the axial position on Mn1 (e.g. Fig. 1, W3) facilitating proton transfers between nonadjacent atoms in the complex. These proton transfer processes may involve formation of bifurcated hydrogen bonds between Glu178 carboxylate and two other donor/acceptor atoms. Two successive proton transfers from a terminally bound peroxide substrate are required for completion of the substrate oxidation half-reaction and reduction of the dimanganese(III) core. In Y42F LPC, one of the intrinsic bases is missing from the catalytic core, blocking the second proton transfer step. In addition, the pKa of the remaining oxo bridge and the redox potentials of the metal centers may be altered in the site containing a single atom bridge, interfering with turnover processes. Insertion of hydroxide at elevated pH to form the bis-atom-bridged cluster in Y42F LPC restores the two-proton reactivity of the WT complex.

The WT and Y42F LPC active sites are also expected to exhibit differences in substrate interactions. Substrate may bind to the ‘open’ form of the oxidized (3,3) Y42F cluster terminally, as predicted for the WT (3,3) complex, or by peroxide insertion in a µ1,1 bridging mode previously predicted for the reduced (2,2) form of the cluster in the WT enzyme. µ-bridging coordination is expected to polarize the O-O bond and favor heterolytic bond cleavage coupled to cluster oxidation. As the (3,3) cluster is already oxidized, the bridging geometry may be unproductive, decreasing the catalytic efficiency of the enzyme, or may even lead to an inactivation pathway involving further oxidation of the cluster to the (4,4) state, contributing to the superoxidized (3,4) component in Y42F. These observations support a mechanism in which the solvent bridges serve as proton storage sites, directing the coordination chemistry of the dimanganese cluster for maximum catalytic efficiency.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References

The staff at the Daresbury Laboratory is acknowledged for operation of the synchrotron facility. Support for this project from the National Institutes of Health (GM 42680 to J.W.W) and BBSRC (GRN 50/B05117 to V.V.B) is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Molecular biology and biochemical preparations
  5. Protein expression
  6. Spectroscopic measurements
  7. X-ray crystallography
  8. Results
  9. Biochemical characterization
  10. X-ray crystal structure of Y42F LPC
  11. Spectroscopic characterization – optical absorption spectroscopy
  12. CD and MCD Spectra
  13. EPR spectra of the (3,3) complex
  14. Discussion
  15. Acknowledgements
  16. References
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