Redox potentials of the blue copper sites of bilirubin oxidases

Spectroelectrochemistry of Redox Enzymes Christenson, Andreas 2006 Link to publication Citation for published version (APA): Christenson, A. (2006). Spectroelectrochemistry of Redox Enzymes. Department of Analytical Chemistry, Lund University. 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L UNDUNI VERS I TY PO Box117 22100L und +46462220000 + model ARTICLE IN PRESS BBABIO-45797; No. of pages: 8; 4C: Biochimica et Biophysica Acta xx (2006) xxx – xxx www.elsevier.com/locate/bbabio Redox potentials of the blue copper sites of bilirubin oxidases Andreas Christenson a , Sergey Shleev a,⁎, Nicolas Mano b,c,⁎, Adam Heller b , Lo Gorton a a b Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Department of Chemical Engineering and the Texas Material Institute, The University of Texas, Austin, TX 78712, USA c Centre de Recherche Paul Pascal-CNRS, Avenue Albert Schweitzer, 33600 Pessac, France Received 25 May 2006; received in revised form 7 August 2006; accepted 21 August 2006 Abstract The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was > 650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV. © 2006 Elsevier B.V. All rights reserved. Keywords: Bilirubin oxidase; Copper enzyme; Redox potential; T1; T2; T3 sites; Redox titration; Spectroelectrochemistry 1. Introduction Bilirubin oxidase BOD (bilirubin:oxygen oxidoreductase, EC 1.3.3.5) is a multi-copper oxidase catalyzing the oxidation of tetrapyrroles, e.g., bilirubin to biliverdin, as well as of diphenols and aryl diamines, by molecular O2, which is reduced to water [1]. The primary structures of BODs from the fungi Myrothecium verrucaria, Pleurotus ostreatus, and Trachyderma tsunodae and from the bacterium (Bacillus subtilis) have been reported (see GenBank website). Of these, the BODs from M. verrucaria and T. tsunodae have been purified and biochemically characterized. The enzymes are glycosylated, have molecular weights of 52–64 kDa and absorb, like other multicopper oxidases, at 600 and 330 nm [2–6]. Though crystallographic data have not yet been published for the BODs, accumulated evidence shows that their catalytic centers comprise four copper ions, classified into three type of sites: type 1 (T1), type 2 (T2), and type 3 (T3) copper ions. This is Abbreviations: BOD, bilirubin oxidase; DET, direct electron transfer; ET, electron transfer; DRT, direct redox titration; MRT, mediated redox titration; Em, midpoint redox potential; Em7, midpoint potential at pH 7.0 ⁎ Corresponding authors. Present address: Nicolas Mano, Department of Chemical Engineering and the Texas Material Institute, The University of Texas, Austin, TX 78712, USA. Tel.: +46 46 222 8191; fax: +46 46 222 4544. E-mail addresses: [email protected] (S. Shleev), also the case for laccase, ascorbate oxidase, and ceruloplasmin [1,7]. In all “blue” multicopper oxidases, including BOD, the T2 and T3 sites form trinuclear clusters, where molecular O2 is reduced to water [1]. The T1 center is the primary site for the oxidation of the electron donating substrate [1,6,8,9]. It absorbs intensely near 600 nm, the transition arising from a Cys S → Cu charge-transfer (CT), which displays a small hyperfine coupling-associated EPR signal [7,8]. The structure of the T1 site has been elucidated from spectral and biochemical data and from sequence analysis [6–12]. The ligands of the T1 copper of M. verrucaria BOD are identical with those of low redox potential multicopper oxidases [7– 9,11,12], i.e., two histidines, a cysteine, and a methionine (see Table 1). The ligands of the T1 copper ions of T. tsunodae BOD are identical with those found in high redox potential laccases (e.g. Trametes versicolor and Trametes hirsuta [6–10,13–15]), i.e., two histidines, a cysteine, and a phenylalanine (see Table 1). According to a recent proposal [16], the BODs from M. verrucaria and T. tsunodae should be classified, respectively, as low and high redox potential multicopper oxidases. As can be seen in Table 1 M. verrucaria BOD has a methionine axial ligand at the T1 site, whereas the axial ligand of the T1 site is phenylalanine in the BOD from T. tsunodae. One previous estimate of the value of the redox potential of the T1 site of M. verrucaria BOD (480 mV [17]) is consistent with the proposed 0005-2728/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2006.08.008 Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008 ARTICLE IN PRESS 2 A. Christenson et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx Table 1 Comparison of the midpoint redox potentials, Em, and the amino acid subsequences for low and high redox potential multicopper oxidases Multicopper Oxidase Subsequence Em, T1 site (mV) High redox Trachyderma tsunodae BOD potential Trametes hirsuta laccase Low redox Myrothecium verrucaria BOD potential Rhus vernicifera laccase Zucchini ascorbate oxidase Human ceruloplasmin (T1A or T1B site) … 474H C H I D F H L E A G F485… GenBank Accession number BAA28668 … 474H C H I D F H L E A G F485… GenBank Accession number Q02497 … 494H C H N L I H A D H D M505 … GenBank Accession numbers BAA02123, BAA03166, B48521, Q12737 … 492H C H F E R H T T E G M503 … GenBank Accession number BAB63411 … 506H C H I E P H L H M G M517 … GenBank Accession number A51027 … 506H C H V T D H I H A G M517 … GenBank Accession number NP_000087 ≥650*(pH 7.0) 780 (pH 6.0) [16,20] ≈ 670* (pH 7.0) 430 (pH 7.5) [41,42] 340 (pH 7.0) [51] 490 (pH 5.5) [43] Notes. “*”—present study, underlined—ligand to the T1 copper; all redox potentials are given vs. NHE. classification [16], but two other studies reported more positive redox potentials for this site (see Table 2) [7,18]. The latter are close to, or even higher than, the redox potential of the T1 site of T. tsunodae BOD (Table 2) [7,18,19]. This distinction was not sufficiently considered in recent studies of the redox states of the T1 site in BODs [7,8,11]. One of our objectives was, therefore, to carefully determine the redox potentials of the T1 sites of both BODs and to reconcile the differences in the reported values. Recently, direct electron transfer (DET) between M. verrucaria BOD and spectrographic graphite was shown to take place under both aerobic and anaerobic conditions [20,21], and the kinetics of M. verrucaria BOD-catalyzed O2 electroreduction in direct electrical contact with carbon electrodes was studied [21,22]. The possibility of electrochemical control of the redox reactions of different multicopper oxidases at electrodes is needed both for fundamental understanding of the basis of biocatalysis and for their applications in biofuel cells. Various electrode materials, including gold, were used in biofuel cells. However, DET between BOD and gold, the focus of this report, has never been reported. High rate DET could be of relevance for electroreduction of O2 to water near neutral pH, for which applications might exist [23–26]. 2. Materials and methods 2.1. Chemicals Na2HPO4, KH2PO4, KCl, NaCl, and K4[Fe(CN)6], all of analytical grade, were obtained from Merck (Darmstadt, Germany). The buffers were prepared using water (18 MΩ) purified with a Milli-Q system (Millipore, Milford, CT, USA). K4[Mo(CN)8] was synthesized and purified as described previously [27]. 2.2. Enzymes BOD from Myrothecium verrucaria was purchased from Sigma (St. Louis, MO, USA). The enzyme was additionally purified to homogeneity as described in [28] and the final specific activity was found to be 35 U per mg of protein. BOD from Trachyderma tsunodae (500 units/mg) was from Amano Enzyme, Inc. (Elgin, IL, USA). One unit of activity is defined as the amount of BOD oxidizing 1 μmol of bilirubin per min at pH 8.4 at 37 °C. The purified BOD preparations were homogeneous as judged from SDS-PAGE [29] and as confirmed by mass-spectrometry. They were stored at − 18 °C until use. The concentration of BOD was determined spectrophotometrically at 600 nm using an ε of 4800 M− 1 cm− 1 [7]. 2.3. Spectroelectrochemical studies The redox potentials of the T1 site of the two BODs were determined by mediated spectroelectrochemical redox titration, MRT, and by direct spectro- electrochemical redox titration, DRT [30–32]. The cell consisted of a 1-cm long gold capillary electrode with an I.D. of 350 μm, serving both as the working electrode and as the cuvette. The input and output optical fibers, respectively FCB-UV 400/050-2 and FC-UV 200, were purchased from Ocean Optics (Dunedin, Florida, USA) and were attached at the ends of the capillary. The system comprised a light source DH-2000, a spectrometer SD 2000 and an analogue to digital conversion board ADC-500 (Ocean Optics). The spectra were recorded with Spectra Win 4.2 software from TOP Sensor System (Eerbreek, The Netherlands). The potential of the gold capillary electrode was controlled by a potentiostat (CV-50W, Bioanalytical Systems, BAS, West Lafayette, IN, USA). Two platinum wires served as counter electrodes and a home-made Ag∣AgCl∣KClsat (197 mV vs. NHE), separated from the enzyme solution by two ceramic frits and a buffer salt bridge, excluding chloride from the enzyme solution, was used as the reference electrode. The potential of the reference electrode was checked before and after each experiment versus a saturated calomel electrode (Hg∣Hg2Cl2∣KClsat;+242 mV vs. NHE) from Radiometer (model K401, Copenhagen, Denmark); its value remained within 1 mV before and after the experiments. The working gold capillary electrode was cleaned for approximately 10 h in freshly prepared 3:1 v/v 96% sulfuric acid, 37% H2O2 Piranha solution (Merck) as described in [15,31,32]. It should be noted that handling of the Piranha solution must occur under the most cautions circumstances. MRT and DRT were carried out according to a previously published protocol for laccase [15]. In the MRTs, initially 50 μM each of the reduced form of the two redox mediators, K4[Mo(CN)8] and K4[Fe(CN)6], was used to enhance the communication between the enzymes and the electrode. It reduces the risk of hysteresis and a shift of the midpoint potential (Em) of the titration curve, due to poor electronic contact, caused by protein insulation and slow heterogeneous electron transfer. A 50 μl aliquot of the BOD solution in 0.1 M phosphate buffer at pH 7.0 also containing the mixture of the two mediators was aspirated through the capillary to replace the buffer of the cell. Em7-values (midpoint potentials at pH 7) were determined by sequentially applying a series of potentials to the gold capillary electrode. Each potential was maintained until the Nernst equilibrium was reached (approximately 5 min) between the oxidized (Ox) and reduced (Red) forms of the mediators, the enzyme, and the poised electrode. The redox mediators were converted stepwise from one redox state to another by changing the applied potential, while the concentrations of the Ox and Red forms of the enzymes were determined from the spectra. Basic titration parameters, such as Em7, b (slope of the titration curve), n (number of electrons), and r (correlation coefficient) were determined from plots of the applied potentials (Eappl) vs. log ([Ox]/[Red]). In the case of DET the experimental procedure was identical to the one for MRT, but the titrations were performed without adding a soluble mediator. Moreover, the measurement time for each applied potential was increased from 5 to 15 min in order to assure that the enzyme redox centers and the electrode reach electrical equilibrium also while the electron path is more resistive. The entire cell and all solutions were deoxygenated by flushing with argon (AGA Gas AB, Sundbyberg, Sweden) before the DRT or MRT experiments. During the redox titrations argon was also flushed through an anaerobic box in which the spectroelectrochemical cell was placed. The K4[Mo(CN)8] solutions were prepared just prior to their use and protected from light to minimize photodecomposition of the oxidized Mo(V) mediator [33]. All reported potentials are referred to NHE and all redox titrations were performed in 0.1 M phosphate buffer at pH 7.0. Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008 ARTICLE IN PRESS A. Christenson et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx 3 Table 2 Comparison of reported parameters of redox titrations of BODs from Trachyderma tsunodae and Myrothecium verrucaria BOD E′m, T1 site (mV) b (mV) n Trachyderma tsunodae 660 (pH 7.0) 90 0.66 615 (pH 6.8) 642 (pH 5.0) 710 (pH 7.0) 76 _ _ 1.5 (0.78) _ 3 (0.71) Mediator system(s); Em (mV) K3 ½MoðCNÞ8 =K4 ½MoðCNÞ8 ; 780 mV K3Fe[(CN)6)]/K4[Fe(CN)6]; 435 mV Co(III)(2,6-PA)2/CoII(2,6-PA)2; 747 mV Co(III)(2,6-PA)2/CoII(2,6-PA)2; 747 mV K3 ½MoðCNÞ8 =K4 ½MoðCNÞ8 ; 780 mV Reference Present study [6] [6] [18] K3Os[(CN)6)]/K4[Os(CN)6]; 640 mV K3 ½WðCNÞ8 =K4 ½WðCNÞ8 ; 520 mV Myrothecium verrucaria 670 (pH 7.0) 74 0.80 K3[Mo(CN)8]/K4[Mo(CN)8]; 780 mV Present study K3 Fe½ðCNÞ6 Þ=K4 ½FeðCNÞ6 ; 435 mV 490 (pH 5.3) _ _ NaI3 =NaI; 536mV or K3 Fe½ðCNÞ6 Þ=K4 ½FeðCNÞ6 ; 433 mV [17] 570 (pH 7.8) _ _ K3 Fe½ðCNÞ6 Þ=K4 ½FeðCNÞ6 ; 435 mV [7] 660 (pH 7.0) 83 3 (0.71) K3 ½MoðCNÞ8 =K4 ½MoðCNÞ8 ; 780 mV [18] K3Os[(CN)6)]/K4[Os(CN)6]; 640 mV K3 ½WðCNÞ8 =K4 ½WðCNÞ8 ; 520 mV Notes. “−”—information not available; in brackets recalculated values based on previously published data are shown; all redox potentials are given vs. NHE. 2.4. Cyclic voltammetry measurements Cyclic voltammograms (CVs) of the BODs on the capillary Au electrodes were recorded using the setup for redox titrations as described previously [15]. CVs of the mediator solutions were obtained with a planar Au electrode (BAS) in a 1 ml electrochemical cell with a Ag|AgCl|3 M NaCl reference electrode (BAS) and a Pt counter electrode. The Au electrode was polished with a DP suspension (0.25 μm; Stuers, Copenhagen, Denmark), followed by an alumina FF slurry (0.1 μm; Stuers), rinsed with Millipore water, and sonicated between and after polishing for 10 min. Then the electrode was kept in concentrated H2SO4 with 10% H2O2 for 1 h, subjected to 30 cycles in 0.5 M H2SO4 and rinsed with Millipore water before use. 3. Results 3.1. Mediated redox titration The Em7-values of the redox couples K3[Mo(CN)8]/K4[Mo (CN)8] and K3Fe[(CN)6)]/K4[Fe(CN)6] were first determined using the spectroelectrochemical cell, then confirmed by cyclic voltammetry at a planar Au electrode. Their values were respectively 780 mV and 435 mV vs. NHE (0.1 M phosphate buffer, pH 7.0, 25 °C) for the K3[Mo(CN)8]/K4[Mo(CN)8] and for the K3[Fe(CN)6)]/K4[Fe(CN)6] couples and did not depend on the direction of the scan (Table 2). The values agree with those previously reported ([33–36] and Table 2). Both couples of the mediators absorb strongly below 500 nm but they are transparent above 500 nm. Next, the Em7-values of the T1 sites of M. verrucaria and T. tsunodae BODs were accurately measured by MRT. Each titration was carried out in both directions, i.e., from the fully oxidized to the fully reduced state of the enzyme (reductive titration) and vice versa (oxidative titration). Typical titration curves of M. verrucaria and T. tsunodae BODs are presented in Figs. 1A and 2A respectively. The spectra of the BODs were recorded at redox equilibrium and spectra of the oxidized, partly reduced, and fully reduced M. verrucaria and T. tsunodae BODs are presented in Figs. 1B and 2B, respectively. Equilibration of the blue copper center at each applied potential was apparent from the stabilization of the absorbance at 600 nm. Because the two redox mediators are transparent above 500 nm, the spectral changes at 600 nm were attributed to the blue copper centers of the fungal BODs. Least-squares linear regression analysis of the 600-nm Nernst plots provided an Em7-value of 670 mV vs. NHE and a slope of 74 mV for M. verrucaria BOD (Fig. 1B, insert; Table 2). Similar calculations for T. tsunodae BOD resulted in an Em7-value of 660 mV vs. NHE and a slope of 90 mV (Fig. 2B, insert; Table 2). The linear correlation coefficients were higher, in both instances, than 0.99. In addition to the least-squares regression analyses, the titration curves were analyzed by direct data fitting [30]. The results are presented in Figs. 1A and 2A for each titration curve. The differences between the reductive and oxidative titration curves were very small, less than 12 mV for both BODs (Figs. 1A and 2A). The enzymes could be reversibly cycled between their fully oxidized and fully reduced states and the Em7-values calculated for the two BODs were practically independent of the direction of the potential scans. However, significant fading of the blue color of the enzymes (up to 10%) after even a single titration cycle (oxidized BOD → reduced BOD → oxidized BOD) was observed for both BODs , i.e., the first and the last Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008 ARTICLE IN PRESS 4 A. Christenson et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx 3.3. Direct (mediatorless) redox titrations Spectroelectrochemical data for solutions containing BOD without mediators and under anaerobic conditions in the Au capillary clearly show that the blue color vanishes when the applied potential is switched from +1000 mV to +50 mV vs. NHE. The fading of the colour can only be explained by the direct reduction of the blue copper sites at the gold capillary electrode. Typical absorbance spectra of the oxidized, partly reduced, and fully reduced forms of M. verrucaria and T. tsunodae BODs in the absence of any mediators are presented in Figs. 3B and 4B, respectively. The redox reactions were reversible and both BODs were reoxidized either by applying an oxidizing potential (Figs. 3 and 4) or by O2 (data not shown). Moreover, the first and the last points of the titrations perfectly coincided with each other (cf. curves 1 and 2 in Figs. 3A and 4A). In contrast, as mentioned above, a significant decrease in the absorbance of the enzymes after titration of both BODs was observed in the MRT experiments (vide supra). Fig. 1. MRT of Myrothecium verrucaria BOD in 0.1 M phosphate buffer, pH 7.0. (A) Potentiometric titration curves (curve 1—oxidative titration, curve 2— reductive titration). (B) Spectra from the titrations, corresponding to oxidized BOD (800 mV), partly reduced BOD (675 mV), and fully reduced BOD (475 mV). Insert: a typical Nernst plot of the dependence of the applied potential (E) versus the absorbance at 600 nm and averaged parameters calculated from the titrations. points of the titrations did not coincide with each other (Figs. 1A and 2A). Moreover, a well-pronounced sigmoidal Nernst plot of the titration in the case of T. tsunodae could be clearly seen (Fig. 2B, insert). Similar, but a less pronounced behavior was observed in the titration curve of the M. verrucaria BOD. 3.2. CV measurements The possibility for DET between M. verrucaria and T. tsunodae BODs and gold under aerobic and anaerobic conditions was investigated using the bare capillary gold electrode. Cyclic voltammograms recorded at sweep rates varying from 1 to 1000 mV s− 1 did not reveal any clear redox transformation of either enzyme in the potential range between − 500 mV and +1000 mV vs. NHE. Changing the pH from 3 to 9 did not lead to the appearance of a clearly traceable faradaic current in the voltammograms. Nevertheless, DET between the bare gold electrode and the copper sites of either of the BODs was confirmed by the much slower, only very low current, spectroelectrochemical measurements (vide infra). Fig. 2. MRT of Trachyderma tsunodae BOD in 0.1 M phosphate buffer, pH 7.0. (A) Potentiometric titration curves (curve 1—oxidative titration, curve 2—reductive titration). (B) Exemplary spectra from the titration corresponding to the oxidized BOD (875 mV), partly reduced BOD (650 mV), and fully reduced BOD (500 mV). Insert: Typical Nernst plot of the applied potential (E) dependence of the absorbance at 600 nm and averaged parameters calculated from the titrations. Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008 ARTICLE IN PRESS A. Christenson et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx 5 evident from the absence of pronounced redox peaks in the CVs that the rate of DET is very low. Even though redox potentiometry is now a routine technique widely used in studies of biological ET processes [37], large discrepancies were reported between the Em-values for identical proteins. The ∼180 mV difference between the reported Emvalues of the T1 sites of the same BOD (Table 2) is an example of such a discrepancy. According to the Nernst equation, b values higher than 59 mV (25 °C) would imply the physically impossible transfer of a fraction of the charge of the electron [37]. The reported slope of 83 mV in the titration curve of M. verrucaria BOD corresponds to an n value of 0.71, and the 76 mV slope in the titration curve of T. tsunodae BOD implies an n value of 0.78 (Table 2). Obviously, these n values, deviating from the expected integers by more that 10%, should be candidates for re-evaluation [38]. Two frequent causes of erroneously low n values (less than 1.0) are incomplete equilibration in the redox titration and/or the presence of multiple potential-wise closely spaced redox couples. The low reported values may well have resulted from the latter (Table 2). As early as in 1970 Wilson and Dutton [37,38] have Fig. 3. DRTof Myrothecium verrucaria BOD in 0.1 M phosphate buffer, pH 7.0. (A) Potentiometric titration curves (curve 1—oxidative titration, curve 2—reductive titration) with two midpoint potentials (E′m) of pronounced ET processes during the reductive titration. (B) Some spectra from the titration corresponding to the oxidized BOD (980 mV), partly reduced BOD (550 mV), and fully reduced BOD (100 mV). The spectroelectrochemical data provide evidence of direct, unmediated heterogeneous ET between both of the BODs and gold. However, the mechanism of this process seems to be more complex than the mechanism of the mediated process. The complexity is reflected in the spectroelectrochemical titration curves (Figs. 3A and 4A). The reductive and oxidative titration curves do not overlap (cf. curves 1 and 2 in Figs. 3A and 4A) and only a single well-pronounced ET process with a low E′mvalue (320 mV) was seen when titrating T. tsunodae BOD (Fig. 4A, curve 1), whereas two ET processes, one in the low and one in the high potential range (460 mV and 805 mV, respectively) were seen in the oxidative titration of M. verrucaria BOD (Fig. 3A). Additionally, the reductive titration curve of M. verrucaria BOD was similar to the oxidative titration curve of T. tsunodae BOD (cf. Figs. 3A and 4A). 4. Discussion The data reveal without any doubt that DET between a gold electrode and both BODs can be established. It is, however, Fig. 4. DRT of Trachyderma tsunodae BOD in 0.1 M phosphate buffer, pH 7.0. (A) Potentiometric titration curves (curve 1—oxidative titration, curve 2—reductive titration) with a midpoint potential (E′m) of pronounced ET processes. (B) Exemplary spectra from the titration corresponding to the oxidized BOD (850 mV), partly reduced BOD (350 mV), and fully reduced BOD (50 mV). Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008 ARTICLE IN PRESS 6 A. Christenson et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx pointed out that two independent, closely spaced one-electron redox pairs can yield a sigmoidal Nernst plot with apparent n values between 0.5 and 1.0. As seen in Figs. 1B and 2B, this could be the case for the MRTs of M. verrucaria and T. tsunodae BOD. As reported by Wilson and Dutton [39,40], the presence of two chemically different cytochrome b constituents results in a sigmoidal Nernst plot yielding an apparent n value close to 0.5. In the present set of experiments the homogeneity of both BODs was confirmed by SDS-PAGE and by mass-spectrometry. Nevertheless cyano-copper “multiforms” of the enzyme can be formed in the presence of cyanide-containing mediators [41], the cyanide changing particularly the coordination of the T2/T3 Cu centers [15]. While this could explain the small difference between the first and the last points of the titration curves observed in the MRT of either BOD, it does not explain the still unacceptable n values obtained from MRTs of T. tsunodae BOD performed using non-cyanide redox couples (Table 2). Furthermore, MRTs with cyano-metal complexes yielded excellent values for laccase and ceruloplasmin [20,35,42,43]. Thus, it is unlikely that the use of cyanide-based mediators is responsible for the scatter in the MRT results of the BODs. With the results shown above one suggestion could be the T. tsunodae BOD has two different T1 sites. Human ceruloplasmin has three different T1 sites, T1A, T1B, and T1 PR with redox potentials of 490 mV, 580 mV, and ∼1000 mV [16]. As shown by Deninum and Vänngård [43], MRT of the T1A and T1B sites of human ceruloplasmin resulted in a well-pronounced sigmoidal Nernst plot. Moreover, their titration curve presented in [43] is very similar to the one we obtain for T. tsunodae MRT (Fig. 2) and the parameters of both redox titrations are also close (n ≈ 0.65, b ≈ 91 mV). This is, however, unlikely to be the case for M. verrucaria BOD, where all previously published primary isoenzyme structures contain only one cysteine residue, a mandatory amino acid for coordination of the T1 copper (GeneBank accession numbers BAA02123, BAA03166, B48521, and Q12737). Additional computer analysis of the primary structure of T. tsunodae BOD shows only one typical T1 site subsequence (C H X; where X is an axial ligand of the T1 site, i.e. F, L, or M) in analogy with all others blue multicopper oxidases (Table 1). Nevertheless, a hypothetical possibility of the presence of a second “abnormal” T1 site in T. tsunodae BOD cannot be ruled out because the enzyme contains five cysteine residues (GenBank Accession number BAA28668). Though that the T2 copper cannot be detected spectrophotometrically and that the bi-nuclear T3 copper only displays a spectral absorbance shoulder near 330 nm, we observe for both BODs changes between 450 and 800 nm (Figs. 3B and 4B). The broad band does not depend on the source of the enzyme and accounts for about 1/5th of the absorbance. Recently, a redabsorbing chromophore, considered to be one of the coppers of the T2/T3 cluster, probably the T2 site, was reported for Trametes hirsuta laccase [15]. Thus, any of several of the chromophores might be responsible for the blue color of the enzyme, and following conclusions can be drawn: Because the extinction coefficients of the individual copper sites are unknown, the redox potentials of the T1 centers of T. tsunodae and M. verrucaria BODs cannot be spectrophotometrically resolved. For this reason, only the lowest value for the T1 site of T. tsunodae BOD is presented (Table 1). Its estimate is based on interpretation of the titration curves of Fig. 2A. Nevertheless, for M. verrucaria BOD, the parameters of the redox titration are those expected for a Nernstian couple and the data agree well with results of Ikeda et al. [18]. Thus, we can consider the new values presented in Tables 1 and 2 to be the best values of the redox potential of the T1 site estimates to date. Recent reviews link the observed high redox potentials of the T1 site, which we also observe, to the methionine-binding of their copper [16,44–46]. The M. verrucaria BOD is an interesting exception to this “rule”. As seen from Figs. 3A and 4A, a pronounced low redox potential ET process is observed during DRTs of both BODs and the middle E′m7-value between the two ET processes is at 390 mV. We proposed, in view of earlier findings [15] that underlying this value is the redox potential of all T2 copper sites of all multicopper oxidases, near 400 mV vs. NHE. The similarity of the titration curves of Figs. 3A and 4A and of previously obtained curves for fungal laccase [15] also points to the fact that one of the redox potentials of the T2/T3 cluster of either of the BODs, most likely the potential of T2, is close to 400 mV. From the applied point of view, non-mediated BOD-based O2 cathodes with Au electrodes would be much too slow. Methods to overcome the sluggish DET include “wiring’ of the BOD in a redox hydrogel [28,47,48], use of conducting nanoparticles, or simply by orientation in thiol based selfassembled monolayers [49,50]. 5. Conclusions The midpoint potential of the T1 site of M. verrucaria BOD is close to 670 mV vs. NHE, whereas a much broader potential range, between 650 and 750 mV, is estimated for the Em7-value of the T1 site of T. tsunodae BOD. A long-range, but a very slow electron transfer between gold and either of the BODs was observed. The earlier suggestion that the redox potentials of the T2 copper sites in all blue multicopper oxidases are similar, i.e. approximately 400 mV, holds for the BODs. Acknowledgment The authors thank the following organizations for financial support: The Swedish Research Council, The Office of Naval Research (N00014-02-1-0144), and the Welch Foundation. The Swedish Institute (SI) is acknowledged for the support of a postdoctoral fellowship for S.S. N.M. thanks The Oronzio de Nora Industrial Electrochemistry Fellowship of The Electrochemical Society. References [1] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Multicopper oxidases and oxygenases, Chem. Rev. 96 (1996) 2563–2605. [2] N. Tanaka, S. Murao, Purification and some properties of bilirubin oxidase of Myrothecium verrucaria MT-1, Agr. Biol. Chem. 46 (1982) 2499–2503. [3] Y. Gotoh, Y. Kondo, H. Kaji, A. Takeda, T. Samejima, Characterization of Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008 ARTICLE IN PRESS A. Christenson et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] copper atoms in bilirubin oxidase by spectroscopic analyses, J. Biochem. (Tokyo) 106 (1989) 621–626. J. Guo, X.X. Liang, P.S. Mo, G.X. Li, Purification and properties of bilirubin oxidase from Myrothecium verrucaria, Appl. Biochem. Biotechnol. 31 (1991) 135–143. S. Koikeda, K. Ando, H. Kaji, T. Inoue, S. Murao, K. Takeuchi, T. Samejima, Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast, J. Biol. Chem. 268 (1993) 18801–18809. J. Hirose, K. Inoue, H. Sakuragi, M. Kikkawa, M. Minakami, T. Morikawa, H. Iwamoto, K. Hiromi, Anions binding to bilirubin oxidase from Trachyderma tsunodae K-2593, Inorg. Chim. Acta 273 (1998) 204–212. A. Shimizu, J.H. Kwon, T. Sasaki, T. Satoh, N. Sakurai, T. Sakurai, S. Yamaguchi, T. Samejima, Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands, Biochemistry 38 (1999) 3034–3042. G. Zoppellaro, N. Sakurai, K. Kataoka, T. Sakurai, The reversible change in the redox state of type I Cu in Myrothecium verrucaria bilirubin oxidase depending on pH, Biosci. Biotechnol. Biochem. 68 (2004) 1998–2000. K. Kataoka, K. Tanaka, Y. Sakai, T. Sakurai, High-level expression of Myrothecium verrucaria bilirubin oxidase in Pichia pastoris, and its facile purification and characterization, Protein Expr. Purif. 41 (2005) 77–83. K. Hiromi, Y. Yamaguchi, Y. Sugiura, H. Iwamoto, J. Hirose, Bilirubin oxidase from Trachyderma tsunodae K-2593, a multi-copper enzyme, Biosci. Biotechnol. Biochem. 56 (1992) 1349–1350. A. Shimizu, T. Sasaki, J.H. Kwon, A. Odaka, T. Satoh, N. Sakurai, T. Sakurai, S. Yamaguchi, T. Samejima, Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties, J. Biochem. (Tokyo) 125 (1999) 662–668. T. Sakurai, L. Zhan, T. Fujita, K. Kataoka, A. Shimizu, T. Samejima, S. Yamaguchi, Authentic and recombinant bilirubin oxidases are in different resting forms, Biosci. Biotechnol. Biochem. 67 (2003) 1157–1159. K. Piontek, M. Antorini, T. Choinowski, Crystal structure of a laccase from the fungus Trametes versicolor at 1.90 Å resolution containing a full complement of coppers, J. Biol. Chem. 277 (2002) 37663–37669. S.V. Shleev, O.V. Morozova, O.V. Nikitina, E.S. Gorshina, T.V. Rusinova, V.A. Serezhenkov, D.S. Burbaev, I.G. Gazaryan, A.I. Yaropolov, Comparison of physico-chemical characteristics of four laccases from different basidiomycetes, Biochimie 86 (2004) 693–703. S. Shleev, A. Christenson, V. Serezhenkov, D. Burbaev, A. Yaropolov, L. Gorton, T. Ruzgas, Electrochemical redox transformations of T1 and T2 copper sites in native Trametes hirsuta laccase at gold electrode, Biochem. J. 385 (2005) 745–754. S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A.I. Yaropolov, J.W. Whittaker, L. Gorton, Direct electron transfer between copper-containing proteins and electrodes, Biosens. Bioelectron. 20 (2005) 2517–2554. F. Xu, W. Shin, S.H. Brown, J.A. Wahleithner, U.M. Sundaram, E.I. Solomon, A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability, Biochim. Biophys. Acta 1292 (1996) 303–311. S. Tsujimura, A. Kuriyama, N. Fujieda, K. Kano, T. Ikeda, Mediated spectroelectrochemical titration of proteins for redox potential measurements by a separator-less one-compartment bulk electrolysis method, Anal. Biochem. 337 (2005) 325–331. T. Ikeda, A novel electrochemical approach to the characterization of oxidoreductase reactions, Chem. Rec. 4 (2004) 192–203. S. Shleev, A. El Kasmi, T. Ruzgas, L. Gorton, Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic graphite electrode, Electrochem. Commun. 6 (2004) 934–939. S. Tsujimura, T. Nakagawa, K. Kano, T. Ikeda, Kinetic study of direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase at carbon electrodes, Electrochemistry (Tokyo) 72 (2004) 437–439. S. Tsujimura, K. Kano, T. Ikeda, Bilirubin oxidase in multiple layers catalyzes four-electron reduction of dioxygen to water without redox mediators, J. Electroanal. Chem. 576 (2005) 113–120. S. Tsujimura, M. Fujita, H. Tatsumi, K. Kano, T. Ikeda, Bioelectrocata- [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] 7 lysis-based dihydrogen/dioxygen fuel cell operating at physiological pH, Phys. Chem. Chem. Phys. 3 (2001) 1331–1335. N. Mano, H.-H. Kim, A. Heller, On the relationship between the characteristics of bilirubin oxidases and O2 cathodes based on their “wiring”, J. Phys. Chem., B 106 (2002) 8842–8848. N. Mano, A. Heller, A miniature membraneless biofuel cell operating at 0.36 V under physiological conditions, J. Electrochem. Soc. 150 (2003) 1136–1138. S. Tsujimura, M. Kawaharada, T. Nakagawa, K. Kano, T. Ikeda, Mediated bioelectrocatalytic O2 reduction to water at highly positive electrode potentials near neutral pH, Electrochem. Commun. 5 (2003) 138–141. C. Gercog, K. Gustav, I. Shtrele, Manual on inorganic synthesis (in Russian), Mir. Moscow (1985) 651–1654. N. Mano, H.-H. Kim, Y. Zhang, A. Heller, An oxygen cathode operating in a physiological solution, J. Am. Chem. Soc. 124 (2002) 6480–6486. A. Chrambach, R.A. Reisfeld, M. Wyckoff, J. Zaccari, A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis, Anal. Biochem. 20 (1967) 150–154. T. Larsson, A. Lindgren, T. Ruzgas, Spectroelectrochemical study of cellobiose dehydrogenase and diaphorase in a thiol-modified gold capillary in the absence of mediators, Bioelectrochemistry 53 (2001) 243–249. N. Bistolas, A. Christenson, T. Ruzgas, C. Jung, F. Scheller, U. Wollenberger, Spectroelectrochemistry of cytochrome P450 cam, Biochem. Biophys. Res. Commun. 314 (2004) 810–816. A. Christenson, E. Dock, L. Gorton, T. Ruzgas, Direct heterogeneous electron transfer of theophylline oxidase, Biosens. Bioelectron. 20 (2004) 176–183. I.M. Kolthoff, W.J. Tomsicek, Oxidation potential of the system potassium molybdocyanide-potassium molybdicyanide, and the effect of neutral salts on the potential, J. Phys. Chem. 40 (1936) 247–255. I.M. Kolthoff, W.J. Tomsicek, The oxidation potential of the system potassium ferrocyanide/potassium ferricyanide at various ionic strengths, J. Phys. Chem. 39 (1935) 945–954. V.T. Taniguchi, B.G. Malmström, F.C. Anson, H.B. Gray, Temperature dependence of the reduction potential of blue copper in fungal laccase, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 3387–3389. J.M. Johnson, H.B. Halsall, W.R. Heineman, Metal complexes as mediator-titrants for electrochemical studies of biological systems, Anal. Biochem. 133 (1983) 186–189. P.L. Dutton, Redox potentiometry: determination of midpoint potentials of oxidation–reduction components of biological electron-transfer systems, Methods Enzymol. 54 (1978) 411–435. G.S. Wilson, Determination of oxidation–reduction potentials, Methods Enzymol. 54 (1978) 396–410. D.F. Wilson, P.L. Dutton, Energy dependent changes in the oxidation– reduction potential of cytochrome b, Biochem. Biophys. Res. Commun. 39 (1970) 59–64. P.L. Dutton, J.B. Jackson, Thermodynamic and kinetic characterization of electron-transfer components in situ in Rhodopseudomonas spheroides and Rhodospirillum rubrum, Eur. J. Biochem. 30 (1972) 495–510. B.R.M. Reinhammar, Oxidation–reduction potentials of the electron acceptors in laccases and stellacyanin, Biochim. Biophys. Acta 275 (1972) 245–259. B. Reinhammar, T.I. Vänngård, Electron-accepting sites in Rhus vernicifera laccase as studied by anaerobic oxidation–reduction titrations, Eur. J. Biochem. 18 (1971) 463–468. J. Deinum, T. Vänngård, The stoichiometry of the paramagnetic copper and the oxidation–reduction potentials of type I copper in human ceruloplasmin, Biochim. Biophys. Acta 310 (1973) 321–330. H.B. Gray, B.G. Malmström, R.J.P. Williams, Copper coordination in blue proteins, J. Biol. Inorg. Chem. 5 (2000) 551–559. H. Li, S.P. Webb, J. Ivanic, J.H. Jensen, Determinants of the relative reduction potentials of type-1 copper sites in proteins, J. Am. Chem. Soc. 126 (2004) 8010–8019. E.I. Solomon, R.K. Szilagyi, S. DeBeer George, L. Basumallick, Electronic structures of metal sites in proteins and models: contributions to function in blue copper proteins, Chem. Rev. 104 (2004) 419–458. Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008 ARTICLE IN PRESS 8 A. Christenson et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx [47] N. Mano, F. Mao, A. Heller, A miniature biofuel cell operating in a physiological buffer, J. Am. Chem. Soc. 124 (2002) 12962–12963. [48] N. Mano, F. Mao, A. Heller, Characteristics of a miniature compartmentless glucose-O2 biofuel cell and its operation in a living plant, J. Am. Chem. Soc. 125 (2003) 6588–6594. [49] D.D. Schlereth, Biosensors based on self-assembled monolayers, Compr. Anal. Chem. 44 (2005) 1–63. [50] U. Wollenberger, Third generation biosensors—integrating recognition and transduction in electrochemical sensors, Compr. Anal. Chem. 44 (2005) 65–130. [51] F. Xu, A.E. Palmer, D.S. Yaver, R.M. Berka, G.A. Gambetta, S.H. Brown, E.I. Solomon, Targeted mutations in a Trametes villosa laccase, axial perturbations of the T1 copper, J. Biol. Chem. 274 (1999) 12372–12375. Please cite this article as: Andreas Christenson et al., Redox potentials of the blue copper sites of bilirubin oxidases, Biochimica et Biophysica Acta (2006), doi:10.1016/j.bbabio.2006.08.008