Spectroscopic and kinetic properties of the horseradish peroxidase mutant T171S Evidence for selective effects on the reduced state of the enzyme Barry D. Howes 1 , Nigel C Brissett 2 , Wendy A. Doyle 2 , Andrew T. Smith 2 and Giulietta Smulevich 1 1 Dipartimento di Chimica, Universita ` di Firenze, Italy 2 Department of Biochemistry, School of Life Sciences, University of Sussex, Brighton, UK Horseradish peroxidase (HRPC) is a member of class III of the plant peroxidase superfamily and is cap- able of utilizing hydrogen peroxide to oxidize a wide range of phenols, anilines and other synthetic sub- strates [1]. Historically, it is has been the subject of extensive spectroscopic and functional studies [2–4] and is the archetypal enzyme on which many of our ideas of biological oxidation reactions have been based [1]. More recently this has involved the detailed characterization of mutants [2–4] designed to probe various aspects of its catalytic mechanism and spectroscopic properties. Detailed structural informa- tion for the enzyme and the catalytic intermediates in all five oxidation states is now available [5]. In contrast to globins, the preferred resting state of per- oxidases is the oxidized ferric state. The structural factors, particularly those in the proximal environ- ment of the haem that underlie the relative stability Keywords Fe-His stretch; haem peroxidase; horseradish peroxidase; resonance Raman; redox potential Correspondence A. T. Smith, Department of Biochemistry, School of Life Sciences, John Maynard Smith Building, University of Sussex, Falmer, Brighton BN1 9QG, UK Fax: +44 1273 678433 Tel: +44 1273 678863 E-mail: A.T.Smith@sussex.ac.uk G. Smulevich, Dipartimento di Chimica, Universita ` di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy Fax: +39 0554573077 Tel: +39 0554573083 E-mail: giulietta.smulevich@unifi.it (Received 17 June 2005, revised 11 August 2005, accepted 30 August 2005) doi:10.1111/j.1742-4658.2005.04943.x Studies on horseradish peroxidase C and other haem peroxidases have been carried out on selected mutants in the distal haem cavity providing insight into the functional importance of the distal residues. Recent work has dem- onstrated that proximal structural features can also exert an important influence in determining the electronic structure of the haem pocket. To extend our understanding of the significance of proximal characteristics in regulating haem properties the proximal Thr171Ser mutant has been con- structed. Thr171 is an important linking residue between the structural proximal Ca 2+ ion and the proximal haem ligand, in particular the methyl group of Thr171 interdigitates with other proximal residues in the core of the enzyme. Although the mutation induces no significant changes to the functional properties of the enzyme, electronic absorption and resonance Raman spectroscopy reveal that it has a highly selective affect on the reduced state of the enzyme, effectively stabilizing it, whilst the electronic properties of the Fe(III) state unchanged and essentially identical to those of the native protein. This results in a significant change in the Fe 2+ ⁄ Fe 3+ redox potential of the mutant. It is concluded that the unusual properties of the Thr171Ser mutant reflect the loss of a structural restraint in the proximal haem pocket that allows ‘slippage’ of the proximal haem ligand, but only in the reduced state. This is a remarkably subtle and specific effect that appears to increase the flexibility of the reduced state of the mutant compared to that of the wild-type protein. Abbreviations ABTS, 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonate); BHA, benzhydroxamic acid; APX, ascorbate peroxidase; CCP, cytochrome c peroxidase; CIP, Coprinus cinereus peroxidase; HRPC, horseradish peroxidase C; LS, low spin; MOPS, 3-morpholinopropanesulfonic acid; TcAPXII, cationic ascorbate peroxidase isoenzyme II from tea; PG, pyrolytic graphite; RR, resonance Raman; 5-c, 5-coordinate; HS, high spin; QS, quantum mechanically mixed spin; SCE, standard calomel electrode; TBMPC, tributylmethyl phosphonium chloride; F221M, Phe221Met mutant HRPC; T171S, Thr171Ser mutant HRPC. 5514 FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS of the ferrous ⁄ ferric state of HRPC, are not under- stood. Spectroscopic and functional studies have concentra- ted predominantly on the role of residues in the distal haem cavity that provide an insight into the role of key catalytic residues [6–9]. A significant outcome of many of these studies [6,10–12] has been the discovery of coupled effects that can be understood on the basis of a hydrogen bonding network that links the distal and proximal halves of the protein. It is perhaps surprising that the proximal domain has received relatively little attention compared to the extensive studies referred to above on the distal cavity. Recent studies, including the removal of the proximal structural Ca 2+ ion [13] and the construction of a mutant in which the proximal Phe221 residue was replaced by Met [14], have demonstrated the ‘sensitivity’ of the proximal region in determining the electronic and structural properties of the haem pocket. In the present study we attempt to obtain further insights into the extent to which the proximal environ- ment influences the electronic and functional properties of the haem. The proximal residue Thr171 that pro- vides two bonds to the proximal Ca 2+ ion and is adja- cent in sequence to the active site residue His170, has been replaced by a serine residue. Ser differs from Thr only by the absence of a methyl group and so repre- sents a very subtle change, a change that is naturally present in other fungal peroxidases belonging to class II, such as lignin peroxidase [3]. This region of the structure has particular relevance in both enzymes because of its potential to provide structural coupling between the proximal Ca 2+ ion and the residues of the active site, most notably the distal His (H170 in HRPC, Fig. 1). The effects of the Thr171Ser mutant have proven to be particularly intriguing and specific to the reduced state of the enzyme. The Fe(II) state of the enzyme has features in common with both the Phe221Met mutant and Ca-depleted proteins whilst the Fe(III) state is essentially identical to that of the wild-type protein. We conclude that the properties of the T171S mutant reflect the loss of a structural restraint in the proximal haem pocket that results in unusually subtle and selective effects that are mediated exclusively on the reduced state of the enzyme. We hypothesize that this residue imposes a degree of rigid- ity to the structure of the reduced state of class III peroxidases. Results and Discussion Table 1 shows some of the functional parameters asso- ciated with the T171S mutant. Its ability to react with hydrogen peroxide to form Compound I, as measured by the second order rate constant for Compound I for- mation, was identical to the wild-type enzyme. Other functional parameters such as steady state turnover with 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulpho- nate) (ABTS) as substrate and the ability to bind the aromatic donor molecule benzhydroxamic acid (BHA), itself a sensitive indicator of the integrity of the distal haem pocket [3,4] were also essentially unaffected. Hence, the mutation causes no gross change in the functional properties of the enzyme and any catalytic consequences of the mutation are at best slight. This finding is strengthened by the electronic absorp- tion and resonance Raman (RR) spectra of the oxid- ized wild-type HRPC and the Thr171Ser mutant (data not shown), which clearly show that mutation does Fig. 1. The structural features of the proximal haem pocket of HRPC showing the haem, the proximal Ca 2+ (grey sphere) H170, and T171. The methyl group of T171S is shown as a green sphere, it can be seen to interdigitate with F172, F229, G168, D230, I228 and is directly constrained by the carbonyl oxygen of G168. Inclu- sion of F221 obscures the local structure in the proximity of T171 and for clarity is omitted. Table 1. Comparison of functional parameters for HRPC proximal pocket mutants. Kinetic parameters were determined as described in the Experimental procedures section. Earlier values for the F221M mutant which stacks against the proximal His (Fig. 1) are shown for comparison. Enzyme variant e a (mM )1 Æcm )1 ) k 1 (M )1 Æs )1 ) Turnover no. (s )1 ) K d (BHA, lM) Recombinant wild-type a 98 ± 3 (1.7 ± 0.1) · 10 7 560 b 2.7 ± 0.3 c F221M a 110 ± 3 (1.5 ± 0.1) · 10 7 510 4.3 ± 0.1 T171S 106 ± 2 (1.6 ± 0.1) · 10 7 607 ± 35 2.5 ± 0.5 c a Values from [14]. b Values from [32]. c Determined in 25 mM Mops pH 7.0 supplemented with 100 lM CaCl 2 . B. D. Howes et al. Selective effects on the reduced state of HRPC FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS 5515 not affect either the coordination or the spin states of the haem system. The Thr171Ser mutant contains a substantially 5-coordinate (5-c) quantum mechanically mixed spin (QS) haem essentially identical to the wild- type protein [4,8,11,15]. Additional evidence that in the oxidized state the structural properties of the HRPC haem cavity are essentially unaffected by the Thr171Ser mutation was obtained from comparison of the electronic absorption and RR spectra of the wild type and Thr171Ser either in the presence of the aromatic donor BHA or at alkaline pH. Addition of saturating amounts of BHA did not reveal any spectral differences between the wild type and the mutant (data not shown). Furthermore, the electronic absorption spectra of the Thr171Ser mutant and wild-type HRPC at pH 10.1 were also identical (data not shown), indicating that the mutant binds a hydroxyl group at alkaline pH, forming a 6-coordinate low spin (LS) haem species in an identical way to the wild type [16,17]. The pK a for the alkaline transition being similar to that of the wild type, % 11.1 [18]. Finally, comparison of the X-ray structures of the oxidized forms of the native [5,19] and the T171S mutant (protein databank code: 1GW2.pdb) did not reveal any significant differences between the two pro- teins. These observations are consistent with the very subtle nature of the mutation, i.e. the loss of a single methyl group, depicted in green in Fig. 1. In marked contrast, for the reduced state compar- ison of the RR spectra of the Thr171Ser mutant and wild type reveal very significant differences. Figures 2 and 3 show the electronic absorption and RR spectra, respectively, of the T171S mutant at pH 6.8 and 8.9 and wild type (pH 6.8) in the Fe(II) state. The previ- ously characterized proximal pocket mutant F221M (pH 6.8) [14] and the Ca-depleted protein (pH 6.8) [13] are also shown for comparison. The electronic absorp- tion spectrum of the T171S mutant is characteristic of a 5-c HS haem, as previously established for the wild- type protein (Fig. 2) [20], while the blue-shift of the Soret and the changes in the visible region of the Ca-depleted and F221M spectra indicate a more or less marked presence of LS haem in these proteins. In agreement with the absorption spectra, the high fre- quency RR spectra of Fe(II) Thr171Ser at pH 6.8 and 8.9 for 441.6 nm excitation (data not shown) are very similar to those of the wild-type enzyme at pH 6.8 [13] and indicative of a 5-c HS haem species. However, the low frequency region of the RR spectrum of the T171S variant differs markedly from that of the parent enzyme. In particular, the wild-type protein is charac- terized by an intense band at 244 cm )1 (Fig. 3), that has been assigned to the m(Fe-Im) stretching mode between the haem iron atom and the imidazole ligand (Im) of the proximal histidine residue [20]. This mode, which is only active in the 5-c HS Fe(II) state in peroxidases is at higher frequencies than found in other haem proteins as a consequence of the strong Fig. 2. Electronic absorption spectra of 40 lM ferrous HRPC. Wild- type protein at pH 6.8 in 25 m M MOPS, T171S at pH 6.8 in 100 m M citrate and pH 8.9 in 100 mM glycine, F221M at pH 6.8 in 100 m M citrate, Ca-depleted at pH 6.8 in 5 mM EDTA, 50 mM Tris ⁄ HCl. The visible region is expanded 8-fold. The path length of the cuvette was 1 mm for all spectra. The ordinate scale refers to the wild-type protein. Wavenumber /cm -1 Fig. 3. Resonance Raman spectra of ferrous HRPC. Buffers as reported in Fig. 2. Experimental conditions: 5 cm )1 resolution; 441.6 nm excitation wavelength; concentration of 50 l M,10s⁄ 0.5 cm )1 collection interval, 20 mW laser power at the sample (wild type, pH 6.8); concentration of 45 l M,12s⁄ 0.5 cm )1 collec- tion interval, 20 mW laser power at the sample (T171S, pH 6.8); concentration of 40 l M,26s⁄ 0.5 cm )1 collection interval, 20 mW laser power at the sample (T171S, pH 8.9); concentration of 70 l M, 5s⁄ 0.5 cm )1 collection interval, 20 mW laser power at the sample (F221M, pH 6.8); concentration of 40 l M,12s⁄ 0.5 cm )1 collection interval, 30 mW laser power at the sample (Ca-depleted, pH 6.8). Selective effects on the reduced state of HRPC B. D. Howes et al. 5516 FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS hydrogen bond between the proximal His170 and Asp247 residues (Fig. 1). This strong hydrogen bond imparts a pronounced imidazolate character to the proximal His [21]. It is evident from Fig. 3 that the RR spectrum of the Thr171Ser mutant at pH 6.8 is very similar to that of the wild-type except for the three bands at 220, 247 and 276 cm )1 , all of which shift to lower frequencies upon raising the pH to 8.9. The pH sensitivity of these frequencies is a common character- istic of all peroxidases and is a consequence of the strong H-bond between the proximal His and Asp resi- dues that is weakened at alkaline pH. Therefore, on the basis of their sensitivity to pH, the bands at 220 and 247 cm )1 are assigned to two m(Fe-Im) modes. The band at 276 cm )1 is assigned to an internal vibra- tional mode of the imidazole ligand, which is enhanced by coupling with the Fe-Im mode, as previously observed for the F221M HRPC mutant [14]. The fre- quencies of the remaining bands in the RR spectrum, which are unaffected by the pH, are assigned by anal- ogy to myoglobin and cytochrome c peroxidase (CCP) [22,23] to out-of-plane modes of the porphyrin ring itself together with the bending modes of the propionyl and vinyl substituents of the haem. Class III peroxidases normally exhibit only one Fe-Im band, in contrast to the class I and II peroxidases that have two Fe-Im bands resulting from the tauto- merism of the imidazole N d proton with respect to the donor and acceptor atoms of the proximal His and Asp H-bond [11]. The only exception to this is the cat- ionic ascorbate peroxidase isoenzyme II from tea (TcAPXII); this shows two Fe-Im stretches at 233 and 249 cm )1 . This is a rather anomalous hybrid peroxi- dase, that exhibits the spectroscopic characteristics and substrate preferences of both class I and class III per- oxidases [24]. As in ascorbate peroxidase (APX) [25], the absence of a decrease of the I 220 ⁄ I 247 intensity ratio between the two bands observed for the Thr171Ser mutant, upon raising the pH, suggests that the two species are independent and not in equilibrium, as is thought to be the case for CCP [23] and Coprinus cine- reus peroxidase (CIP) [26]. The frequencies of the two m(Fe-Im) stretching modes at 220 cm )1 (downshifted 24 cm )1 compared to wild-type) and 247 cm )1 (up shif- ted 3 cm )1 compared to wild-type) are very close to those found for the F221M mutant. These are distin- guished by the strength of the hydrogen bond between the proximal His and the Asp carboxylate side chain. In structural terms, these observations could be related to changes in the steric constraints operating at the proximal His and ⁄ or Asp residues induced by the T171S mutation. It appears as though the C a back- bone in the His170 region may be more mobile due to the absence of the methyl group at position 171, T171 presumably normally constrains any ‘slippage’ of the adjacent His170 (Fig. 1). Interestingly, the introduction of this flexibility into the proximal cavity structure leads to two populations of molecules. In the first case the Fe-Im bond strength is decreased (band at 220 cm )1 ), this is similar to the situation that arises upon removal of the proximal structural calcium ion (217 cm )1 ) [13]. In the second case the opposite effect is seen, which is much less pronounced (band at 247 cm )1 ). The redox potential for the Thr171Ser mutant (E ¼ )32 ± 7 mV vs. SCE) was determined to be significantly less negative than that of the wild-type (E ¼ )133 ± 7 mV vs. SCE). The increase in the redox potential compared to the wild type is in accord with the observation of a m(Fe-Im) mode in Thr171Ser at a markedly lower frequency than in the wild-type protein (220 cm )1 ) (Fig. 3). In fact, a greater imidazolate character, stabilizing the higher oxidation state, leads to a decrease of the redox potential of the heme iron. However, it is not pos- sible to make a direct correlation between the magni- tude of the changes in Fe-His band frequencies and the redox potential values. This is exemplified by the case of CCP and its mutants D235E, D235N and D235A [27]. The H-bond between the proximal His and Asp235 is completely lost when Asp235 is replaced by the nonbonding residues Asn and Ala, but the D235E mutation results only in a very small displacement of the carboxylate group. Nevertheless, in all three cases the RR frequency of the Fe-His band is at 205 cm -1 (CCP wild type, 246 ⁄ 233 cm -1 ), suggesting that in the three mutants the proton is no longer shared between residue 235 and His175. How- ever, the redox potentials of the mutants increase compared to wild type by 70 (D235E), 104 (D235N) and 105 (D235A) mV. The redox potential depends on a number of other factors such as the electro- static, Van der Waals and hydration status of the haem environment that also vary with the peroxidase under consideration, while the Fe-His frequency is primarily dependent on the strength of the Fe-His bond and hence on the status of the proximal His H-bond. It appears that one can readily rationalize the general trends but not the magnitude of the chan- ges seen. Entropy factors can in principle play an important part in determining the redox potential of HRPC. In fact, contrary to the situation found for electron transfer proteins, reduction of HRPC leads to a marked increase in entropy [28]. Thus, the greater flexibility of the proximal cavity structure evident in B. D. Howes et al. Selective effects on the reduced state of HRPC FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS 5517 the ferrous state of the mutant may contribute to an increase in the disorder and hence entropy of the reduced state of the mutant compared to that of the wild-type protein. In contrast to the present study, in previous cases where the proximal site of HRPC has been modified by mutation [14] or Ca-depletion [13] significant chan- ges in the properties of the ferric form of the protein has been detected. Even so, the changes detected in the haem cavity of the reduced state appear more promin- ent. In both cases significant structural alterations to the protein conformation were indicated, not only by marked changes in the geometric disposition of the proximal His and Asp residues, affecting the imidazo- late character of the His, but also by the formation of a LS species. The latter indicating the probable bind- ing of His42 to the haem iron, i.e. a major collapse or rearrangement of the distal cavity has taken place. Hence, the overall conclusion that may be drawn is that modification of the proximal cavity of HRPC by mutation or Ca 2+ ion removal has a significant impact on the properties of His170. The strength of the hydro- gen bond between the proximal His and Asp residues, and thus the imidazolate character of the His is expec- ted to modulate not only the strength of the Fe-Im bond but also the stability of the different oxidation states. In fact, the potential sensitivity and dependence of enzyme properties on the structural characteristics of the proximal domain is demonstrated by the mark- edly less negative (by approximately 100 mV) redox potential of the T171S mutant compared to the wild- type protein. Mutations of distal residues can give rise to a sub- stantial reduction of the catalytic activity if they have a direct impact on the disposition of the catalytically important His42 and Arg38 residues [29,30]. However, although the Fe-Im bands of many distal mutants undergo a small but significant shift to lower frequen- cies compared to the wild-type protein, their redox potentials are virtually unaffected [30,31]. It is appar- ent that the effects resulting from proximal changes in HRPC, particularly those affecting the Ca 2+ ion site are far reaching. This underlines the importance of long range interactions originating form the prox- imal cavity in fine tuning the properties of the haem, most notably the haem iron redox potential. The most significant finding in this study is the effect of a single mutation on the structural constraints of the protein, whereby a relatively minor alteration to the proximal cavity is capable of selectively stabilizing the reduced state of the enzyme but is having essen- tially no detectable affect on the oxidized form of the enzyme. Experimental procedures Site-directed mutagenesis and expression of recombinant proteins A PCR-based method was used for site-directed mutagenesis that utilized the synthetic HRP C gene [32] exactly as des- cribed in [14]. The construction of the Thr171Ser mutant involved the use of the pSD18 template. Oligonucleotide primer WDHRP9 (5¢-GAGTGTCCGGAGGCCACAGCT TTGG-3¢; where mutated bases are shown in bold) was designed for the point mutation at position 171 and to over- lap the BspEI site. WDHRP10 (5¢-CATAGGGATCCTT ATTAAGAGTTGC-3¢) was designed to overlap the BamHI site at the 3¢ end of the gene. A mutant DNA insert (430 bp) was generated by PCR. The purified fragment was inserted into the cloning vector pBGS19 via ‘blunt-ended’ ligation and checked by automated DNA sequencing (Applied Bio- systems, Foster City, CA, USA). Only the expected muta- tion was detected. The plasmid insert was digested using BspEI and BamHI and ligated in frame into pSD18 [32] cut with the same restriction enzymes. The whole HRPC insert was then excised from pSD18 with NdeI and BamHI and ligated into the expression vector pFLAG1 at the unique NdeI and BglII sites. Expression of HRPC in E. coli W31110, isolation of inclusion bodies, refolding and purification of the wild-type protein and the Phe221Met and Thr171Ser mutants were carried out as previously described [14,32,33]. Purified recombinant enzyme Thr171Ser was stored at )80 °Casa frozen solution in 10 mm Mops buffer at pH 7.0. Steady-state turnover with 2,2¢-azinobis-(3-ethyl- benzothiazoline-6-sulphonate) (ABTS) Peroxidase activity was determined in 50 mm phos- phate ⁄ citrate buffer pH 5.0 at 25 °C, by measuring the increase in absorbance at 405 nm given by the formation of the 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) cation radical product with 1.5 mm H 2 O 2 and 0.3 mm ABTS as described in [32]. Measurement of the second-order rate constants for Compound I formation The rate of Compound I formation (k 1 ) was determined in 10 mm sodium phosphate buffer pH 7.0 (l ¼ 100 mm) and 25 °C under pseudo-first-order conditions (Applied Photo- physics SX18MV stopped-flow system; Leatherhead, UK) by following the decrease in absorbance at 395 nm. Time courses were fitted to single exponentials and the rate constants (k obs ) determined from the fits. Values for k obs were plotted against H 2 O 2 concentration and linear pseudo- first order plots were obtained over the substrate range studied. The k 1 value was obtained from the gradient. Selective effects on the reduced state of HRPC B. D. Howes et al. 5518 FEBS Journal 272 (2005) 5514–5521 ª 2005 FEBS Determination of dissociation constants for benzhydroxamic acid The dissociation constants (K d ) of complexes formed between resting state enzymes and benzhydroxamic acid were determined by titration of the Soret region of the vis- ible spectrum as described previously [33]. K d values were calculated by fitting the data to Eqn. (1) using a weighted least squares error minimization procedure. A ¼ 2A 1 L=fðL þ K d þ PÞþ½ðL þ K d þ PÞ 2 À 4PL 1=2 gð1Þ The absorbance change at 408 nm resulting from benzhydr- oxamic acid of concentration L, binding to a total protein concentration P, was determined, while allowing the remaining K d and maximum absorbance change at satura- tion (A 1 ) to float. Resonance Raman and electronic absorption spectroscopy For resonance Raman and electronic absorption spectro- scopy the experimental conditions were as reported in the captions to figures. Samples of ferrous enzymes for electronic absorption and resonance Raman spectroscopy were pre- pared by addition of 2 lL of dithionite (20 mgÆmL )1 )to 50 lL of deoxygenated peroxidase solution. Benzhydroxamic acid complexes were prepared by adding aliquots of 0.2 m benzhydroxamic acid (Sigma, St Louis, MO, USA) in 10 mm MOPS pH 7.0 to the enzyme samples, to a final (saturating) concentration of 5 mm. Electronic absorption spectra, measured with a Cary 5 spectrophotometer, were recorded both prior to and after RR measurements. No degradation was observed under the experimental conditions used. RR spectra were obtained at room temperature with excitation from the 406.7 nm line of aKr + laser (Coherent, Innova 90 ⁄ K, Santa Clara, CA, USA), and from the 441.6 nm line of a HeCd laser (Liconix, xxxx, xxxx). The back-scattered light from a slowly rotating NMR tube was collected and focused into a computer-controlled double monochromator (Jobin-Yvon HG2S, xxxx, xxxx) equipped with a cooled photomultiplier (RCA C31034A, xxxx, xxxx) and photon counting electro- nics. To minimize local heating of the protein by the laser beam, the sample was cooled by a gentle flow of N 2 gas passed through liquid N 2 . RR spectra were calibrated to an accuracy of 1 cm )1 for intense isolated bands, with indene as the standard for the high-frequency region and with indene and CCl 4 for the low-frequency region. Redox potential measurements The redox potential measurements were made by firstly embedding the protein in a tributylmethyl phosphonium chloride (TBMPC) membrane followed by immobilization on a pyrolytic graphite (PG) electrode surface as previously described [34]. DC cyclic voltammograms were run in previ- ously degassed 0.1 m sodium phosphate, pH 7.0. Measure- ments were carried out at 25 °C in a glass microcell (sample volume, 1 mL). During the measurements the anaerobic environment was maintained by a gentle flow of high-purity grade nitrogen just above the surface of the solution. A PG electrode (AMEL, Milan, Italy) was the working electrode, a saturated calomel electrode (AMEL) was the reference and a Pt ring the counter-electrode. An Amel 433 ⁄ W multipolarograph (Milan, Italy) interfaced with a PC as data processor was employed for voltammet- ric measurements. The potentials reported in the text are referenced to the standard calomel electrode (SCE). The redox potential of the wild-type protein determined by this method is approximately 100 mV less negative than that determined using potentiometry [30,34]. However, differ- ences of this order between the values of the redox poten- tial of proteins measured using cyclic voltammetry and potentiometry have been noted previously [35]. Acknowledgements This work was supported by the EU Biotechnology Programme, ‘Towards Designer Peroxidases’ BIO4- CT97-2031 (to G.S. and A.T.S.), Italian CNR and ex 60% (to G.S) and the BBSRC under B17590 to A.T.S. The authors acknowledge the COST action D21 ‘Met- allo Enzymes and Chemical Biomimetics’ for support- ing the exchange among the different laboratories. The authors are grateful to Prof. R. Santucci for carrying out the redox potential measurements. References 1 Dunford HB (1999) Heme Peroxidases, 1st edn. Wiley- VCH, New York. 2 Veitch NC & Smith AT (2001) Horseradish peroxidase. Adv Inorg Chem 51, 107–162. 3 Smith AT & Veitch NC (1998) Substrate binding and catalysis in heme peroxidases. Curr Opin Struct Biol 2, 269–278. 4 Veitch NC (2004) Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry 65, 249–240. 5 Berglund GI, Carlsson GH, Smith AT, Szo ¨ ke H, Henriksen A & Hajdu J (2002) The catalytic pathway of horseradish peroxidase at high resolution. 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