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J. Phys. Chem. B 2007, 111, 3488-3495
Redox Characteristics of a de Novo Quinone Protein Sam Hay,† Kristina Westerlund,† and Cecilia Tommos*,†,‡ Department of Biochemistry & Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm UniVersity, SE-106 91 Stockholm, Sweden, and Department of Biochemistry & Biophysics, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104-6059 ReceiVed: October 10, 2006; In Final Form: January 18, 2007
The electrochemistry of 2,6-dimethylbenzoquinone (DMBQ) has been characterized for three different systems: DMBQ freely solvated in aqueous buffer; DMBQ bound to a neutral, blocked cysteine (N-acetylL-cysteine methyl ester) and the resulting DMBQ-bCys compound solvated in aqueous buffer; and DMBQ bound to a small model protein denoted R3C. The goal of this study is to detect and characterize differences in the redox properties of the protein-ligated DMBQ relative to the solvated quinones. The R3C protein used here is a tryptophan-32 to cysteine-32 variant of the structurally defined R3W de novo protein (Dai et al. J. Am. Chem. Soc. 2002, 124, 10952-10953). The properties of R3C were recently described (Hay et al. Biochemistry 2005, 44, 11891-11902). DMBQ was covalently bound to bCys and R3C through a sulfur substitution reaction with the cysteine thiol. In contrast to the solvated DMBQ and DMBQ-bCys compounds, diffusion controlled electrochemistry of DMBQ-R3C showed well-behaved and fully reversible n ) 2 oxidation/reduction with a peak separation of ∼30 mV between pH 5 and 9. DMBQ-R3C could also be immobilized on a gold electrode modified with a self-assembled monolayer of 3-mercaptopropionoic acid, allowing the measurement, by cyclic voltammetry, of an apparent rate of electron transfer of 22 s-1. The (cysteine) sulfur substitution significantly lowers one of the hydroquinone pKA’s from 10.4 in DMBQ to 6.8 in DMBQ-bCys. This pKA is slightly elevated in DMBQ-R3C to 7.0 and the E1/2 at pH 7.0 is raised by 110 mV from +190 mV in DMBQ-bCys to +297 mV in DMBQ-R3C.
Introduction Quinones are a common type of cofactor found in enzymes and redox proteins. They can be noncovalently bound, as is the caseofthequinonesfoundintherespiratory1-3 andphotosynthetic4-6 electron-transfer chains as well as in the pyrroloquinoline quinone (PQQ) containing proteins,7 or covalently bound, as is the case of the post-translationally generated cofactors such as tryptophan tryptophylquinone (TTQ) found in bacterial dehydrogenases8,9 and 2,4,5-trihydroxyphenylalanine quinone (TPQ) in the copper amine oxidases.8,10 General structural motifs for the binding sites of non-protein-derived quinones are emerging.11 The best-characterized binding site is the QA site of the photosynthetic reaction centers from purple bacteria,4-6 which recently has been shown to be structurally similar to the QA site of photosystem II12,13 and to have similarities to the quinone QD binding site in the E. coli fumarate reductase.1,14 Quinone binding is stabilized by hydrogen bonding to the quinone carbonyl/hydroxyl groups, van der Waals contact between the protein and the quinone aromatic head group, and especially by hydrophobic interactions to the large quinone phytol tail.15 The protein environment plays a major role in modulating the thermodynamic properties of the quinone cofactor and the kinetics of the electron- and proton-transfer reactions associated with its redox chemistry (e.g., refs 2, 6, and 16-18). As an example, in the type II photosynthetic reactions centers (photosystem II and the reaction centers from purple bacteria) the * To whom correspondence should be addressed. E-mail address:
[email protected]. Telephone: 215-746-2444. Facsimile: 215573-7290. † Stockholm University. ‡ University of Pennsylvania.
final two electron acceptors are quinones. The penultimate acceptor, the primary quinone QA, is only involved in oneelectron reduction and is not doubly reduced under physiological conditions. This contrasts with the ultimate acceptor, the secondary quinone QB, which is reduced in two separate oneelectron reductions while concertedly or subsequently becoming protonated. Differences in the protein environment can (at least qualitatively) account for the differing redox properties of QA and QB in the bacterial systems. QA is tightly bound within a relatively hydrophobic part of the M subunit while QB is loosely associated with a more polar region of the L subunit. The semiquinone anions can be stabilized by a positive electrostatic potential, which can be provided through hydrogen bonding, by charged neighboring amino acids, and by the dipole of the protein backbone. The backbone dipole may play a significant role, as it is unusually large in reaction centers.19 Despite the essential role of quinones in a myriad of biochemical redox reactions, there are very few examples in the literature of electrochemical characterization of the proteinassociated quinone cofactors beyond simple potentiometric measurements. Typically the large size of quinone-containing proteins, and the fact that many of them are membrane bound, has hampered voltammetry measurements. This is in contrast with the wide range of reports describing the voltammetry characteristics of these cofactors either free in solution20-24 or covalently attached to electrodes (e.g., refs 25 and 26). A few studies have been performed on quinones in natural proteins in which different strategies have been used to acquire the voltammetry data. Fujieda et al. characterized the redox properties of the cysteine tryptophylquinone (CTQ) in Paracoccus denitrificans amine dehydrogenase by spectroelectrochemistry
10.1021/jp066654x CCC: $37.00 © 2007 American Chemical Society Published on Web 03/10/2007
CV Characterization of a Model Quinone Protein and cyclic voltammetry (CV).27 The CTQ cofactor of this enzyme is located in the smallest γ subunit of the Rβγ heterotrimeric protein complex and, while no electrochemical response could be detected for the intact system, the isolated γ subunit gave rise to a pair of anodic and cathodic waves in the cyclic voltammogram. Kong et al. resolved the electrochemical response from two bacteriochlorophyll species and QA of the Rhodobacter sphaeroides reaction center by CV and square wave voltammetry.28 Rapid and reversible electron exchange between the cofactors and the electrode was achieved by preparing specific protein film samples containing either lipid vesicle or polyions. Gray and co-workers synthesized an electrode attached covalently bridged “wire” designed to bind in the substrate channel of the Arthrobacter globiformis amine oxidase and successfully established rapid electron tunneling to and from the deeply buried TPQ site of this enzyme.29 Recently, Haehnel and co-workers reported the construction and characterization, including cyclic voltammetry traces, of templateassembled four-helix bundles containing covalently attached quinones.30 Here we complement and extend these protein quinone studies by reporting the CV characteristics of a quinone bound to a small model protein denoted R3C. This protein is a Trp-32 to cysteine variant of the structurally defined R3W de novo protein,31 which originally was designed to study the redox properties of aromatic amino acids.32,33 In an earlier study, three different mercaptophenols were bound to R3C via disulfide linkage to the buried Cys-32 residue.34 The CD and NMR spectral characteristics of the three mercaptophenol-R3C proteins were shown to be overall very similar to those of R3C and R3W, and it was concluded that the phenol ligation did not give rise to any major structural changes in the three-helix bundle scaffold. This conclusion has recently been reinforced by more detailed NMR studies on the mercaptophenol-R3C proteins (Tommos et al., unpublished data). In the present study, we have ligated 2,6-dimethyl-1,4-benzoquinone (DMBQ) to the unique and buried Cys-32 in R3C via a 1,4-Michael-type thiol addition reaction. It has long been known that unsaturated quinones such as p-benzoquinone form adducts with thiol-containing compounds35 including cysteine.36 2,3-Dimethoxy-5-methyl-1,4benzoquinone(CoQ0)and2-methyl-1,4-naphthoquinone(menaquinone) have been shown to bind to a surface-exposed cysteine of human oxyhemoglobin37,38 and more recently CoQ0, menaquinone and 2,3,5-trimethyl-1,4-benzoquinone were bound to a surface-exposed cysteine on cytochrome c39 and to a cysteine engineered into a synthetic four-helix bundle.30 Additionally, one of the authors in this present work recently bound a variety of p-benzoquinones to an engineered cysteine in cytochrome b562.40 In this study, DMBQ was chosen as it is relatively small and hydrophobic and is thus predicted to have a minimized impact on the protein structure if it is incorporated within the hydrophobic interior of the protein. DMBQ was also chosen so that there is only one possible isomer of the cysteine-reacted species and that the position para to the sulfur bond would be occupied. This latter point is important as the para-carbon in sulfur-substituted quinones is activated toward nucleophilic attack and readily becomes substituted with a hydroxide group.41,42 As noted in our recent study on mercaptophenols bound to R3C,34 the main focus of our model-protein approach is not on the absolute redox properties of the ligated cofactors but rather on how these properties may change when the aromatic molecules are bound to the protein relative to being freely solvated in aqueous buffer. Accordingly, in addition to investigating the redox characteristics of DMBQ-R3C by CV,
J. Phys. Chem. B, Vol. 111, No. 13, 2007 3489 equivalent studies were performed on DMBQ and on DMBQ substituted with the charge-neutral cysteine analogue N-acetylL-cysteine methyl ester. Experimental Methods Materials. 2,6-Dimethyl-1,4-benzoquinone (DMBQ), guanidine hydrochloride (Gdn:HCl), N-acetyl-L-cysteine methyl ester (bCys), 3-mercaptopropionoic acid (MPA), trifluoroacetic acid (TFA), and thrombin were purchased from Sigma. 5,5′Dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) was purchased from Aldrich and dithiothreitol (DTT) from Saveen & Werner AB. Preparation of DMBQ-r3C Samples. A gene coding for R3C was cloned into a modified Novagen pET32 vector and coexpressed with thioredoxin in E. coli BL21(DE3)pLysS cells. Following cell lysis, the thioredoxin-R3C fusion protein was separated from the cytosolic fraction on a nickel column and digested with thrombin. R3C was isolated from the digestion mixture via a second nickel-column step followed by reversed phase HPLC using a C18 semipreparative (Grace Vydac) column and a acetonitrile/water/TFA solvent system. Due to the thrombin cleavage site, a GS N-terminal extension (numbered here as -2 and -1) is added to the R3C sequence. The resulting 67-residue polypeptide chain has the following sequence: (GS-RVKALEEKVKALEEKVKALGGGGRIEELKKKCEELKKKIEELGGGGEVKKVEEEVKKLEEEIKKL). Binding of the aromatic cofactor to R3C was performed essentially as described in ref 34 with the following modifications: Lyophilized R3C was dissolved in 50 mM potassium phosphate (KPi), 3 M Gdn:HCl, pH 8.0. A ∼5-fold excess of DTT was added and the sample gently agitated for 3 h at room temperature. Following a gel filtration buffer exchange step into 50 mM KPi, pH 7.0, the protein concentration was determined by the Ellman’s assay under denaturing conditions (6 M Gdn: HCl) and using an 412 of 13.7 mM-1 cm-1. A 5-fold excess of DMBQ was reacted with the reduced R3C protein in 50 mM KPi, 3.2 M Gdn:HCl, pH 7.0. The sample was subsequently dialyzed against a 50 mM KPi pH 7.0 buffer containing quinhydrone (E° of 298 mV at pH 7.0) and ferricyanide (E° of 436 mV at pH 7.0) at a 1:1 and 1:5 protein to oxidant mole ratio, respectively. The DMBQ-binding step and the dialysis were carried out overnight at 4 °C in the dark. The aim of the dialysis step was to maximize the concentration of oxidized DMBQ-R3C and to simplify the HPLC purification of this species from the reaction mixture. The dialyzed sample was buffer exchanged into 50 mM sodium acetate, pH 5.5, and the oxidized DMBQ-R3C protein was then isolated on an analytical C18 reversed phase HPLC column using a 30-70% acetonitrile over 80 min acetonitrile/water/TFA gradient (0.50% acetonitrile/ min, 1 mL/min flow rate). With this gradient, the oxidized form of DMBQ-R3C eluted at 40% acetonitrile. The elution of DMBQ-R3C was followed by monitoring the absorbance at 220 (protein backbone and DMBQ absorbance), 262 (oxidized DMBQ-R3C absorbance), and 305 nm (reduced DMBQ-R3C absorbance). Following the C18 step, the protein samples were lyophilized and stored at -20 °C. The yield of DMBQ-R3C was about 25% relative to the starting R3C material. The R3C and DMBQ-R3C preparations were pure as assayed by gel electrophoresis and their molecular weights verified by electrospray ionization mass spectrometry. The calculated and experimental molecular weights of the two proteins are listed in Table 1. Preparation of DMBQ-bCys Samples. Nine milligrams of N-acetyl-L-cysteine methyl ester (bCys) was reacted overnight
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TABLE 1: Protein Properties of DMBQ-r3C and r3Ca molecular mass calcd molecular mass measd 273 % helix ∆GH2O m
DMBQ-R3C
R3C
7595.4 7595 ( 1 10600 ( 900 65 ( 2%;c 60 ( 1%d -2.7 ( 0.2 1.7 ( 0.1
7460.2 7460 ( 1 ∼200b 72%b -4.0 ( 0.1 2.0 ( 0.1
a Units are as follows: molecular mass, Da; 273, M-1 cm-1; ∆GH2O kcal mol-1; m, kcal mol-1 M-1. b From ref 34. c pH range 4.3-5.9, Figure S3. d pH range 5.9-10.3, Figure S3.
at room temperature with 25 mg of DMBQ dissolved in a 8 mL of 50 mM KPi pH 7.0 and 50% ethanol solution. A 3-4fold excess of DMBQ over bCys was used to reoxidize the bCys-bound hydroquinone formed during the reaction and to prevent the formation of DMBQ-(bCys)2. The DMBQ-bCys product was purified from the reaction mixture on a C18 semipreparative reversed phase column using a 20-40% acetonitrile over 31 min acetonitrile/water/TFA acid gradient (0.65% acetonitrile/min, 5 mL/min flow rate). The purification was followed by monitoring the absorbance at 200, 300, and 400 nm. DMBQ eluted in 24% acetonitrile, while oxidized DMBQ-bCys eluted in 27% acetonitrile. A second species was resolved, which had an absorbance spectrum consistent with the reduced hydroquinone form of DMBQ-bCys, but was discarded. Following purification, the DMBQ-bCys samples were lyophilized and stored at -20 °C. Electrochemistry. Measurements were performed with an Autolab PGSTAT12 potentiostat equipped with a conventional three-electrode glass cell containing a 2 mm diameter gold working electrode, a platinum wire counter electrode, and a Ag/ AgCl (3.0 M NaCl) reference electrode. The glass cell and the electrodes were purchased from Princeton Applied Research (Gammadata, Uppsala, Sweden) except the Pt wire counter electrode, which was from Bioanalytical Systems (West Lafayette, IN). All potentials quoted given here are relative to the normal hydrogen electrode (NHE). The effects of uncompensated resistance were minimized by using the Positive Feedback iR Compensation function available in the Autolab GPES software package. Values used (typically ∼50-800 Ω) were just below those at which current oscillations were observed.43 The gold electrodes were treated as follows: The electrode surface was polished with an alumina slurry, rinsed with water, and sonicated. Following this, the surface was electrochemically cleaned in 100 mM H2SO4 by cycling 10 times between 0.4 and 0.95 V and then 15 times between 0.4 and 1.55 V. The scan rate was 0.1 V s-1, and the scans started and ended at 0.6 V. The clean electrode was rinsed with water followed by ethanol and then immersed in a freshly prepared solution of 5 mM MPA in ethanol for at least 30 min. The electrode was rinsed with ethanol followed by water and air-dried. At this stage, the electrode was either used for electrochemical measurements or immediately immersed in a 4 °C, 5 mM KPi pH 7.0 solution of protein to prepare a monolayer. All solutions for electrochemistry were prepared with milli-Q water, and all measurements were made at room temperature under an argon atmosphere. The peak positions and peak heights of each voltammogram were determined with the GPES software. When necessary, voltammograms were Fourier-filtered to remove highfrequency noise, and the background current was removed by subtracting a linear baseline (see Supporting Information Figure S4).
Results and Discussion UV-vis Spectra of DMBQ-r3C and Reference Compounds. The absorption spectra of oxidized DMBQ, DMBQbCys, and DMBQ-R3C are shown in Figure 1A, and the spectra of the reduced species are shown in Figure 1B. The absorption maxima of the oxidized and reduced states are also listed in the Supporting Information (Table S1). The spectrum of oxidized DMBQ has absorption maxima at 257 and around 336 nm, while the reduced hydroquinone species absorbs maximally at 253 nm. The absorptions bands of the oxidized quinone at 257 and 336 nm arise due to π f π* transitions.44 The spectrum of aqueous DMBQ clearly changes upon the addition of bCys, and there are also distinct spectral differences between the solvated DMBQ-bCys compound and DMBQ bound to the buried Cys-32 residue of the R3C protein. The spectrum of oxidized DMBQ-bCys displays two pronounced absorption maxima at 240 and 264 nm, a shoulder around 300 nm, and a broad absorption band around 430 nm, while the reduced state gives rise to a well-resolved peak centered at 305 nm. Sulfur substitution of 1,4-benzoquinone causes the second (lower energy) π f π* transition to red shift41 and the absorbance of DMBQ-bCys at 264 is likely to represent the second π f π* transition, red-shifting from 257 nm in DMBQ. The spectrum of oxidized DMBQ-R3C exhibits a weak shoulder around 273 nm while reduced DMBQ-R3C displays an absorption maximum around 307 nm. The line shape of the oxidized DMBQR3C spectrum is much less distinct relative to the spectra of the freely solvated quinone compounds, which is partly due to the spectral overlap between the bound quinone and the protein backbone. However, we note that the R3C scaffold has no significant absorbance at 273 nm (273 ∼ 200 M-1 cm-1),34 and thus the absorbance at 273 nm represents the ligated quinone. In order to estimate an extinction coefficient for oxidized DMBQ-R3C at 273 nm, the well-characterized R3W protein was used as a reference. The protein concentration of a R3W sample was determined optically by assuming a 280 nm extinction coefficient of 5690 M-1 cm-1 for Trp-32.32 The protein concentration of a DMBQ-R3C sample was then determined by using the R3W sample as the standard in a BioRad protein concentration assay. The R3W standard curve was found to be linear up to a protein concentration of 0.4 µM (OD595 e 0.28), and consequently this range was used to determine the concentration of a freshly prepared DMBQ-R3C sample. Both proteins were dissolved in a 10 mM sodium acetate, pH 5.5, 15 mM KCl buffer and the data recorded using a Varian Cary 400 double monochromator spectrometer. From these measurements, we estimate that 273 equals 10600 ( 900 M-1 cm-1 for the oxidized form of DMBQ-R3C (Table 1). Protein characteristics of DMBQ-r3C. The content of the secondary structures and the global stability of DMBQ-R3C were examined by circular dichroism (CD) spectroscopy. R3W was used as a reference also for these measurements. Earlier studies have established that the R3W CD spectrum shows no significant change over a pH 4-10 range32 and that its 222 nm amplitude represents a 76% R-helical protein.31 The far-UV CD spectra of the R3W and DMBQ-R3C samples described above are shown in Figure 1C. The line shape of the two spectra are very similar and show the characteristic feature of a predominantly R-helical protein with the double minima at 222 and 208 nm. By comparison of the amplitude at 222 nm for the two CD spectra, the DMBQ-R3C protein is estimated to be 65% R-helical at pH 5.5. This value is close to the 72% helical content of R3C (Table 1),34 suggesting that the binding of DMBQ does not significantly alter the R-helical content of the
CV Characterization of a Model Quinone Protein
J. Phys. Chem. B, Vol. 111, No. 13, 2007 3491
Figure 1. (A) Normalized UV-vis spectra of air-oxidized DMBQ (green), DMBQ-bCys (blue), and DMBQ-R3C (black) dissolved in 10 mM sodium acetate, pH 5.5. (B) Normalized UV-vis spectra of reduced DMBQ (green), DMBQ-bCys (blue), and DMBQ-R3C (black) dissolved in 10 mM sodium acetate, pH 5.5, containing 3 mM sodium dithionite. The spectra were collected with a Varian Cary 400 spectrometer at room temperature. (C) Far-UV circular dichroism spectra of R3W (red) and DMBQ-R3C (black) dissolved in 15 mM KCl, 10 mM sodium acetate, pH 5.5. The spectra were collected with a Jasco J-720 CD spectrometer at room temperature.
Figure 2. (A) Cyclic voltammograms of DMBQ at pH 6.0 (a) and pH 11.0 (b) and of DMBQ-bCys at pH 6.0 (c). All samples were in 10 mM KPi and measured at a scan rate of 50 mV s-1. (B) Cyclic voltammogram of DMBQ-R3C on an Au-MPA electrode in 5 mM KPi at pH 7.0 (a) and pH 6.0 (b) measured at a scan rate of 10 mV s-1. (C) Half-wave potential E1/2 as a function of solution pH for DMBQ (open squares), DMBQ-bCys (filled triangles), and DMBQ-R3C (open circles). The solid lines represent fits to eq 1.
R3C scaffold. This result is consistent with our earlier study, which showed that 2-, 3-, and 4-mercaptophenol could be ligated to Cys-32 in R3C without any major changes in the R-helical content of the protein.34 The pH stability of DMBQ-R3C was also assessed by CD spectroscopy by monitoring the 222 nm amplitude between pH 4.3 and 10.3 (Supporting Information Figure S1). The helical content was found to be 65 ( 2% between pH 4.3 and 5.9 and slightly lowered to 60 ( 1% between pH 5.9 and 10.3 (Table 1). The effect of DMBQ binding on the global stability of R3C was examined by monitoring the loss of secondary structures as a function of guanidine:HCl (Gdn:HCl) concentration. Chemical denaturation plots of R3C and DMBQ-R3C obtained at pH 5.5 (data not shown) were fitted using the method described by Santoro and Bolen,45 and the derived free energy of unfolding, ∆GH2O, and the m values are given in Table 1. The stabilities of R3C and DMBQ-R3C are -4.0 ( 0.1 and -2.7 ( 0.1 kcal mol-1, respectively. As a comparison, the stability of R3W is 3.3 ( 0.1.34 Thus DMBQ-R3C is stable in aqueous solution and remains helical over a broad pH range. As described earlier, R3C is a single residue variant of R3W and the solution structure of the latter predicts that the cysteine is essentially completely buried in R3C. It is thus likely that bound quinone is partly solvent exposed and partly shielded by the protein matrix. The drop in stability between R3C and DMBQ-R3C might reflect the presence of a quinone oxygen atom in the hydrophobic core of protein. If the Cys-32 ligated quinone would be fully solvent exposed and reside on the surface of the protein, the core packing of the three-helix bundle must change quite substantially and we would anticipate a more significant perturbation of the helical content and the stability than is observed. Moreover, the clear differences observed in the optical properties of DMBQ-R3C relative to DMBQ-bCys suggest that the envi-
ronment of the protein-ligated DMBQ is different from the fully solvated DMBQ-bCys compound. Electrochemistry of DMBQ-r3C and Reference Compounds. All cyclic voltammetry measurements described here were performed at room temperature in argon-purged KPi buffer using a 3 mm gold working electrode modified with a selfassembled monolayer of 3-mercaptopropionoic acid (MPA). Below we start by describing the CV characteristics of the two solution reference compounds at these conditions and then focus on the DMBQ-R3C system. Cyclic Voltammetry of DMBQ. Unlike more hydrophobic quinones, DMBQ is sufficiently soluble in water to allow its electrochemical characterization without the presence of an organic solvent. The addition of KCl as a supporting electrolyte however significantly reduces the solubility of DMBQ and was avoided. Additionally, the concentration of DMBQ was kept below 1 mM. Due to the lack of KCl in the sample buffer, the CV scan rate was kept below 100 mV s-1 to avoid errors due to uncompensated internal resistance. The CV characteristics of DMBQ on an MPA-Au electrode were investigated in 10 mM KPi over a pH 2.9-13.3 range and using a scan rate of 50 mV s-1. Two typical voltammograms from this pH study are shown in Figure 2A in which traces a and b represent oxidation/reduction of DMBQ at pH 6.0 and pH 11.0, respectively. The difference between the anodic (oxidative) and the cathodic (reductive) peak potentials, ∆Ep ) |Epa - Epc|, varied considerably as a function of pH with an overall trend of decreasing at alkaline pH (Supporting Information, Figure S2, panel A). For example, ∆Ep equals 554 mV at pH 4.0, 366 mV at pH 7.0, and 70 mV at pH 13.0. The ratio of the anodic and cathodic peak currents, ipa/ipc, also displays a significant pH dependence with an overall trend of approaching unity at the very limits of the investigated pH range, e.g., ipa/ipc
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TABLE 2: Electrochemical Properties of DMBQ, DMBQ-bCys, and DMBQ-r3Ca E1/2 (pH 7) mV vs NHE
E1/2 (pH 0)a mV vs NHE
pK(1)a (QH-/QH2)
pK(2)a (Q2-/QH-)
+179 +190 +297
+592 ( 4 +587 ( 8 +698 ( 1
10.4 ( 0.30 6.84 ( 0.09 6.98 ( 0.08
12.6 ( 0.6 b b
DMBQ DMBQ-bCys DMBQ-R3C
a The parameters are obtained from a fit of eq 1 to the data shown in Figure 2C. b Not determined.
equals 0.79 at pH 2.9, 0.42 at pH 4.0, 0.57 at pH 7.0, and 0.87 at pH 13.0. The width at half-height of the anodic and cathodic waves, δa and δc, was 172 ( 27 and 213 ( 69 mV, respectively, over the whole pH 2.9-13.3 range. The observed quasireversible waveforms with broad, widely separated peaks are characteristic of the proton-coupled redox reactions of quinones in aqueous solution.24 The cyclic voltammogram represents an overall n ) 2 transition in which the potentials of the individual quinone redox couples, the proton affinities of the hydroquinone hydroxyl groups relative to the solution pH, and the rate of the electron transfers and coupled protonic reactions are reflected in the waveform. Typically, peak potentials representing solvated quinones change as a function of scan rate and do not represent equilibrium potentials. Nonetheless, the pH dependence of the peak potentials can be used to gain information on pKA values and reaction pathways involved in the oxidation-reduction processes.24,46,47 DMBQ E1/2 vs pH. The change in half-wave potential, E1/2 ) (Epc + Epa)/2, as a function of solution pH was determined to probe the pKA values of the hydro-DMBQ. A E1/2 vs pH diagram is shown in Figure 2C (open squares) in which the displayed DMBQ data are fitted to46
E1/2(pH) ) E1/2(pH0) +
ln(10)RT/2F log
[(
)(
)]
10-pK(1) + 10-pH 10-pK(2) + 10-pH 1 + 10-pK(1)
1 + 10-pK(2)
(1)
with ln(10)RT/F fixed to 59 mV and pK(1) and pK(2) assigned to the two pKA values of the hydroquinone QH- + H+ T QH2 and Q2- + H+ T QH- equilibria, respectively. This model assumes that the quinone redox behavior is n ) 2 over the whole pH range measured, which is not entirely valid at the pH extremes. Nonetheless, by fitting the DMBQ data in Figure 2C to eq 1 we derive pKA values of 10.4 ( 0.3 and 12.6 ( 0.6 (Table 2), which are in excellent agreement with the pKA values of 10.35 and 12.4 obtained for DMBQ by optical fast-flow methods.48 The MPA-Au electrode has a net negative charge at neutral pH (electrochemical titration estimates the surface pKA of an MPA monolayer to 5.2 ( 0.1).49 Both ipa and ipc systematically decreased above pH 10 until they were no longer resolved at pH g 13.3. This is shown in Figure S2, panel B, in the Supporting Information and can be explained by the electrostatic repulsion between the negatively charged MPA monolayer and the deprotonated hydroquinone. Despite this, the MPA-Au electrode does not significantly perturb the observed reduction potential of DMBQ as the 179 mV half-wave potential of DMBQ measured at pH 7.0 (E1/2 (pH 7.0); Table 2) is in good agreement with literature values of 160-180 mV for dimethylbenzoquinones in aqueous solvents.46,50 Cyclic Voltammetry of DMBQ-bCys. The redox properties of the solution reference DMBQ-bCys compound were investigated under the same conditions as described above for DMBQ, with the exception that the pH range spanned from 3.1 to 9.5. Trace c in Figure 2A represents a cyclic voltammo-
gram of DMBQ-bCys recorded at pH 6.0. The shape and position of the anodic and cathodic waves obtained from DMBQ-bCys are very similar to those representing DMBQ at pH 6.0 (Figure 2A, trace a). A large peak separation was observed over the whole pH range with decreasing values at increasing pH, e.g., ∆Ep was 497 mV at pH 4.0, 408 mV at pH 7.0, and 364 mV at pH 9.0. ipa/ipc was ∼0.6 in the pH 3.1-7.0 range and then rapidly declined at more alkaline conditions. δa and δc were 136 ( 20 and 171 ( 32 mV, respectively, over the investigated pH range. DMBQ-bcys E1/2 vs pH. The pH dependence of the DMBQ-bCys E1/2 value is shown in Figure 2C (filled triangles). Linear fits of the DMBQ-bCys E1/2 values at pH below and above 7.0 have slopes close to 59 mV and 29 mV/decade, respectively (data not shown). A pH dependence of 59 mV/ decade at room temperature is indicative of a one electron/one proton or a two electron/two proton redox reaction, while a pH dependence of 29 mV/decade only arises due to a two electron/ one proton reaction. Thus at pH < 7 the redox reaction is Q + 2e- + 2H+ T QH2 and at pH > 7 the reaction is Q + 2e- + H+ T QH-. To estimate the pKA of the hydroquinone QH- + H+ T QH2 equilibrium more closely, the DMBQ-bCys data in Figure 2C were fitted by eq 1 yielding a pK(QH-/QH2) value of 6.84 ( 0.09 (Table 2). Thus the bCys substitution lowers the DMBQ pK(QH-/QH2) value by about 3.5 pKA units. This increase in acidity probably arises from the more positive inductive effect of sulfur relative to hydrogen, which is predicted to stabilize the deprotonated states of the hydroquinone. The electrochemical response of DMBQ-bCys rapidly declined at pH above 9.5, so unfortunately the pKA representing the Q2+ H+ T QH- equilibrium could not be obtained. Typically, pK(Q2-/QH-) - pK(QH-/QH2) is around 2 for hydroquinones in water,47 which predicts a pK(Q2-/QH) of about 9 for DMBQ-bCys. The bCys data show a linear pH dependence at pK(QH-/QH2) < pH e ∼9.5 putting a lower limit on pK(Q2-/ QH-) of about 9.5. In the DMBQ-bCys E1/2 vs pH fit displayed in Figure 2C, the pK(Q2-/QH-) value has been fixed to 9.5, although we note that this is a predicted and not a measured value. Consistent with a lower pK(Q2-/QH-) value in the mercapto-substituted compound, decline in the electrochemical signal starts at lower pH for bCys-DMBQ relative to DMBQ. Either the increasingly poorer DMBQ-bCys response at pH above 9.5 arises from electrostatic repulsion between the deprotonated hydroquinone and the electrode surface or the mercapto-substituted quinone compound decomposes at high pH. The DMBQ and DMBQ-bCys E1/2 values are very close in the pH range below the pK(QH-/QH2) of the latter compound. Thus, fitting the two model compound data sets in Figure 2C with eq 1 gives E1/2 (pH 0) values that are not significantly different: DMBQ, 592 ( 4 mV and DMBQ-bCys, 587 ( 8 mV. The minor effects on the Q/QH2 potential upon sulfur addition are consistent with earlier studies on mercaptosubstituted benzoquinones.39-41 In the alkaline region the DMBQ-bCys potential is more oxidizing than the DMBQ potential, due to the substantial reduction in the hydroquinone pK(QH-/QH2) upon bCys substitution of the aromatic ring. The DMBQ and DMBQ-bCys E1/2 values at pH 0 and pH 7.0 are listed in Table 2. The DMBQ-bCys voltammogram showed the presence a second redox-active species, whose anodic peak is marked with a star in Figure 2A. The voltammogram recorded at pH 3.1 fully resolved the anodic and cathodic waves of this species (shown in the Supporting Information, Figure S3). The con-
CV Characterization of a Model Quinone Protein taminating species appears to undergo a two-electron reduction (∆Ep ∼ 30 mV) and had a 58 ( 2 mV/decade pH dependence (pH 3.1-5.5) with E1/2 (pH 0) ) 505 ( 8 mV. Above pH 5.5 the cathodic peak was not resolved from the DMBQ-bCys cathodic peak, but the anodic peak maintained this pH dependence (52 ( 1 mV/decade) up to pH 11.5. As for the origin of this second species, the E vs pH diagram of p-benzoquinone becomes complicated by reversible addition of OH- ions at pH > pK(Q2-/QH-).51 It is possible that the redox-active species observed here may be such a hydroxide adduct of DMBQbCys; however it is unlikely that this species would show such a narrow ∆Ep value. Another possibility is that a fraction of DMBQ-bCys is forming a monolayer on the electrode. The DMBQ-bCys sample did not maintain a useful waveform over a sufficiently large range of scan rates to determine the scan rate (V) dependence of this species (i ∝ V1/2 vs i ∝ V), and the origin of the contaminating species was not investigated further. Cyclic Voltammetry of DMBQ-r3C. The redox characteristics of DMBQ-R3C on an MPA-Au electrode was investigated as a function of pH and as a function of scan rate. For the protein measurements, the addition of KCl was also avoided since the salt interfered with the protein/electrode interactions and lowered the electrochemical response of DMBQ-R3C. Two voltammograms are shown in Figure 2B in which traces a and b represent DMBQ-R3C in 5 mM KPi at pH 7.0 and 6.0, respectively, and using a scan rate of 10 mV s-1 (see Supporting Information Figure S4, top panel, for a CV buffer trace of the MPA-modified gold electrode). We have shown earlier that apoR3C is redox inert,34 and thus the observed Faradaic current is due to the reduction and oxidation of the quinone bound to R3C. The two DMBQ-R3C traces are distinctly different from the data described above for the two quinones free in solution (Figure 2A) and are more in line with the electrochemical response of the three natural quinoproteins studied previously,27-29 which all showed fairly narrow peak widths and small peak separations. Notably, the interactions between the protein and the electrode surface are distinctly different for the two DMBQR3C traces shown in Figure 2B. At pH 7.0 the electrochemistry was confirmed to be a surface-confined reaction by the linear scan rate (ν) dependence of ipa and ipc (see Supporting Information, Figure S5), which have slopes of 315 ( 9 and 500 ( 19 nA (V s-1)-1, respectively (R2 both >0.99). The electroactive surface coverage was estimated by integration of the peak current to be ∼25 pmol cm-2,52 which is consistent with a moderately packed monolayer of a small protein. The E1/2 (pH 7.0) value is 286 mV, and the cyclic voltammogram exhibits a ∆Ep of 16 mV. Theoretically, the peak separation is predicted to be 0 V for thin-layer systems at low scan rates, although this is typically not observed for protein films.53 Upon lowering the pH of the electrode buffer to 6.0 or raising it to 8.0, the electrochemistry became diffusion-controlled as judged by a linear dependence of the Faradaic current vs V1/2 (Figure S5). When the solution pH was returned to 7.0 the voltammogram did not return to the waveform of the surfaceconfined protein originally recorded at pH 7 and the E1/2 (pH 7.0) value is 11 mV more oxidizing than the electrode-bound quinone. Thus, the protein/electrode interactions are clearly sensitive to both the pH and the preparation method. Despite this DMBQ-R3C maintains a good diffusion-controlled response, as described in more detail below. As the DMBQR3C protein is only 7.5 kDa and thus much smaller than most natural proteins that are studied by thin-layer voltammetry, the strength of the electrostatic interaction between the protein and the electrode, which is related to both charge and surface area,
J. Phys. Chem. B, Vol. 111, No. 13, 2007 3493 is expected to be quite weak. At pH 8.1, the solution pH approaches the isoelectric point of the protein (calculated to be 9.2 for apo-R3C) resulting in the protein scaffold becoming progressively more neutrally charged. At acidic pH, the MPA carboxylates will begin to protonate, similarly disrupting the electrostatic binding of the protein to the electrode. The diffusion-controlled electrochemistry displays the following characteristics at pH 6.0 (Figure 2B, trace b), 7.0, and 8.1 (data not shown): The peak separation at V ) 10 mV s-1 is 25, 39, and 38 mV at pH 6.0, 7.0, and 8.1, respectively. Thus, the peak separation in the DMBQ-R3C voltammograms is reduced by 1 order of magnitude relative to the CV traces obtained on the two DMBQ solution systems described above and is close to the expected ∆Ep of 30 mV for a diffusioncontrolled two-electron oxidation-reduction process at room temperature. The ratio of the peak currents was below 1.0 at all pH values measured. At pH < 7, ipa/ipc ∼ 0.40, increasing above pH 7 to 0.62 at pH 8.6. Likewise, δa and δc are broader than expected for a diffusion-controlled n ) 2 redox reaction. At pH < 7, δa is 76 mV and increases to 89 mV at pH 8.6. Similarly, δc is 83 mV at pH 5.6 and increases to 110 mV at pH > 7. The nonunity ipa/ipc ratio and the broadened δ values are characteristic for quasi-reversible redox systems such as proton-coupled systems and may arise due to a breakdown of the n ) 2 cooperativity. DMBQ-r3C E1/2 vs pH. The E1/2 value for DMBQ-R3C was determined between pH 4.8 and 8.6, as shown in Figure 2C (open circles). In a similar manner as for the DMBQ-bCys data, linear fits of the DMBQ-R3C E1/2 values at pH < 6.5 and pH > 7.3 have slopes of 58 ( 2 and 28 ( 2 mV/decade, respectively. Fitting the data with eq 1 provided a pK(1) of 6.98 ( 0.08, and again we assign this value to the QH- + H+ T QH2 equilibrium of protein-bound DMBQ (Table 2). The pH dependence of the DMBQ-R3C E1/2 was not determined above pH 8.6 as the protein did not maintain a good interaction with the electrode at alkaline pH. This is consistent with an pK(1) value of 7, which predicts a poor contact between DMBQR3C and the Au-MPA electrode at alkaline pH due to the negative charge of the deprotonated hydroquinone. Consequently, the pK(Q2-/QH-) value for DMBQ-R3C could not be measured. Although the pK(1) of DMBQ is not significantly changed upon binding to the protein (relative to DMBQ-bCys), the E1/2 values are clearly increased. Fitting the DMBQ-R3C E1/2 vs pH data in Figure 2C with eq 1, provided an E1/2 (pH 0) of 698 ( 1 mV and an E1/2 (pH 7.0) of 297 mV (Table 2). The halfwave potential of the protein-bound DMBQ is thus more oxidizing by 110-120 mV, relative to DMBQ and DMBQbCys. DMBQ-r3C Electron-Transfer Kinetics. It is possible that the measured E1/2 of DMBQ-R3C is influenced by slow electron tunneling between the surface of the working electrode and the protein-bound quinone. To investigate this further, the CV waveform representing the DMBQ-R3C monolayer pH 7.0 sample was characterized as a function of scan rate. The results are displayed in Figure 3 as a trumpet plot in which the positions of Epa and Epc are plotted vs the scan rate V.54 Representative cyclic voltammograms of DMBQ-R3C recorded at V ) 10 mV s-1 and 9.6 V s-1 are shown in the Supporting Information (Figure S4). The peak potentials are independent of V at rates below 40 mV s-1 and then split symmetrically around E1/2 ) (Epc + Epa)/2 at higher rates. Over the range of scan rates analyzed (3 mV to 100 V s-1), E1/2 remains constant at 286 (
3494 J. Phys. Chem. B, Vol. 111, No. 13, 2007
Figure 3. “Trumpet plot” of the Epa, Epc (circles), and E1/2 (diamonds) values for DMBQ-R3C immobilized on an Au-MPA electrode in 5 mM KPi, pH 7.0. The E1/2 data are fit with a straight line, and the Ep data (for V g 2.5 V s-1) are fit to eq 2.
4 mV. Thus the measured E1/2 value is not governed by electrontransfer kinetics and appears to be a true equilibrium value. The apparent rate of electron transfer between the electrode and the DMBQ-R3C monolayer at pH 7 was determined by the method of Lavirion.55,56 The Epc and Epa peak positions at V g 2.5 V s-1 were fit to the following equations
Epc ) E - (RT/RnF) ln[R/(RTk/nFV)] 0
Epa ) E0 + (RT/(1 - R)nF) ln[(1 - R)/(RTk/nFV)] (2) with the transfer coefficient, R, fixed at 0.5, and E0 ) E1/2 at Vminimum ) 0.286 V. The fits are shown in Figure 3 and values for n and k, the heterogeneous electron-transfer rate constant, are n ) 2.0 ( 0.1, ka ) 28 ( 4 s-1, and kc ) 16 ( 3 s-1. The value of n ) 2.0 is in excellent agreement with the proposed concerted two-electron mechanism proposed for this system. The average electron-transfer rate of 22 ( 6 s-1 is much slower than the reported electron-transfer rate of cytochrome c on the same type of electrode (MPA-modified Au), which is ∼2 × 103 s-1.57 This difference in rate is not surprising as the redox chemistry of a heme vs a quinone is quite different. The difference in the electron-transfer rates could also be influenced by variations in the coupling between the proteins and the electrode. Conclusions In this study we describe differences in the redox properties of protein-ligated 2,6-dimethylbenzoquinone relative to the solvated quinone species. DMBQ, DMBQ thioether conjugated with N-acetyl-L-cysteine methyl ester (bCys), and DMBQ thioether conjugated to R3C were characterized by cyclic voltammetry using a 3-mercaptopropionoic acid modified gold electrode. The interactions between DMBQ-R3C and the surface of the electrode were pH dependent, and both diffusioncontrolled and thin layer data could be obtained. DMBQ, DMBQ-bCys, and DMBQ-R3C all exhibit nonunity ipa/ipc ratios and broadened peak widths, which are characteristic for quasi-reversible redox systems such as proton-coupled systems. The two small-molecule systems give rise to cyclic voltammetry waveforms with widely separated anodic and cathodic peaks (Figure 2A). In contrast, DMBQ-R3C voltammograms display ∆Ep values close to expected 30 mV for a diffusion controlled two-electron oxidation-reduction process at room temperature (Figure 2B). Sulfur (cysteine) addition to DMBQ lowers the pKA of the QH- + H+ T QH2 equilibrium by about 3 orders of magnitude. This pKA is only slightly increased when DMBQ is bound to R3C relative to bCys (Figure 2C, Table 2). Thus,
Hay et al. the pKA of QH-/QH2 is dominated by the sulfur rather than by the protein. The diffusion controlled E1/2 of DMBQ-R3C at pH 7.0 is +297 mV (Figure 2C and Table 2) and the monolayer E1/2 is +286 mV (Figure 2B). Thus, monolayer formation does not significantly alter the observed reduction potential. The rate of electron transfer between the immobilized quinone protein and the electrode is quite slow (22 ( 6 s-1, Figure 3), yet the observed E1/2 value appears to be a true equilibrium value. Finally, the binding of DMBQ to R3C increases the observed E1/2 at pH 7 by ∼110 mV relative to DMBQ-bCys (Figure 2C). In order to connect structural motifs to the electrochemical data presented here, as well as to the data presented in our earlier study on phenols bound R3C, more detailed structural information is required. NMR spectroscopy efforts to obtain such data are now in process. Acknowledgment. This work was funded by the Swedish Research Council and the Carl Trygger and Magn. Bergwall Foundations. S.H. was supported by a postdoctoral fellowship from the Wenner-Gren Foundation (Stiftelsen Wenner-Grenska Samfundet). Supporting Information Available: A table of quinone absorbance maxima, a graph of changes in the helical content as a function of pH, graphs showing electrochemistry and peak potential as a function of pH, cyclic voltammograms of DMBQ-bCys and DMBQ-R3C, and graphs of scan rate vs ipc for DMBQ-R3C cyclic voltammograms obtained at pH 7.0, 6.0, and 8.1. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Cecchini, G.; Maklashina, E.; Yankovskaya, V.; Iverson, T. M.; Iwata, S. FEBS Lett. 2003, 545, 31-38. (2) Osyczka, A.; Moser, C. C.; Dutton, P. L. Trends Biochem. Sci. 2005, 30, 176-182. (3) Ohnishi, T.; Salerno, J. C. FEBS Lett. 2005, 579, 4555-4561. (4) Allen, J. P.; Williams, J. C. FEBS Lett. 1998, 438, 5-9. (5) Heathcote, P.; Fyfe, P. K.; Jones, M. R. Trends Biochem. Sci. 2002, 27, 79-87. (6) Wraight, C. A. Frontiers Biosci. 2004, 9, 309-337. (7) Anthony, C. Arch. Biochem. Biophys. 2004, 428, 2-9. (8) Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 1476614768. (9) Davidson, V. L. AdV. Protein Chem. 2001, 58, 95-140. (10) Mure, M. Acc. Chem. Res. 2004, 37, 131-139. (11) Fisher, N.; Rich, P. R. J. Mol. Biol. 2000, 296, 1153-1162. (12) Ferreira, K. N.; Iverson, T. M.; Maghlaoui, K.; Barber, J.; Iwata, S. Science 2004, 303, 1831-1838. (13) Loll, B.; Kern, J.; Saenger, W.; Zouni, A.; Biesiadka, J. Nature 2005, 438, 1040-1044. (14) Iverson, T. M.; Luna-Chavez, C.; Cecchini, G.; Rees, D. C. Science 1999, 284, 1961-1966. (15) Warncke, K.; Gunner, M. R.; Braun, B. S.; Gu, L. Q.; Yu, C.-A.; Bruce, J. M.; Dutton, P. L. Biochemistry, 1994, 33, 7830-7841. (16) Ivancich, A.; Artz, K.; Williams, J. C.; Allen, J. P.; Mattioli, T. A. Biochemistry 1998, 37, 11812-11820. (17) Rinyu, L.; Martin, E. W.; Takahashi, E.; Maroti, P.; Wraight, C. A. Biochim. Biophys. Acta 2004, 1655, 93-101. (18) Zhu, Z. Y.; Gunner, M. R. Biochemistry 2005, 44, 82-96. (19) Gunner, M. R.; Nicholls, A.; Honig, B. J. Phys. Chem. 1996, 100, 4277-4291. (20) Mure, M.; Klinman, J. P. J. Am. Chem. Soc. 1993, 115, 71177127. (21) Itoh, S.; Ogino, M.; Haranou, S.; Terasaka, T.; Ando, T.; Komatsu, M.; Ohshiro, Y.; Fukuzumi, S.; Kano, K.; Takagi, K.; Ikeda, T. J. Am. Chem. Soc. 1995, 117, 1485-1493. (22) Mure, M.; Wang, S. X.; Klinman, J. P. J. Am. Chem. Soc. 2003, 125, 6113-6125. (23) Murakami, Y.; Tachi, Y.; Itoh, S. Eur. J. Org. Chem. 2004, 30743079. (24) Rich, P. R. Biochim. Biophys. Acta 2004, 1658, 165-171. (25) Katz, E.; Schlereth, D. D.; Schmidt, H.-L. J. Electroanal. Chem. 1994, 367, 59-70.
CV Characterization of a Model Quinone Protein (26) Sato, Y.; Fujita, M.; Mizutani, F.; Uosaki, K. J. Electroanal. Chem. 1996, 409, 145-154. (27) Fujieda, N.; Mori, M.; Kano, K.; Ikeda, T. Biochemistry 2002, 41, 13736-13743. (28) Kong, J. L.; Lu, Z. Q.; Lvov, Y. M.; Desamero, R. Z. B.; Frank, H. A.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 7371-7372. (29) Hess, C. R.; Juda, G. A.; Dooley, D. M.; Amii, R. N.; Hill, M. G.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 2003, 125, 7156-7157. (30) Li, W.-W.; Hellwig, P.; Ritter, M.; Haehnel, W. Chem. Eur. J. 2006, 12, 7236-7245. (31) Dai, Q.-H.; Tommos, C.; Fuentes, E. J.; Blomberg, M. R. A.; Dutton, P. L.; Wand, A. J. J. Am. Chem. Soc. 2002, 124, 10952-10953. (32) Tommos, C.; Skalicky, J. J.; Pilloud, D. L.; Wand, A. J.; Dutton, P. L. Biochemistry 1999, 38, 9495-9507. (33) Westerlund, K.; Berry, B. W.; Privett, H. K.; Tommos, C. Biochim. Biophys. Acta 2005, 1707, 103-116. (34) Hay, S.; Westerlund, K.; Tommos, C. Biochemistry 2005, 44, 11891-11902. (35) Snell, J. M.; Weissberger, A. J. Am. Chem. Soc. 1939, 61, 450453. (36) Redfearn, E. R. In Biochemistry of quinones; Morton, R. A., Ed.; Academic: New York, 1965; pp 149-181. (37) Winterbourn, C. C.; French, J. K.; Claridge, R. F. C. Biochem. J. 1979, 179, 665-673. (38) Guo, Q.; Corbett, J. T.; Yue, G.-H.; Fann, Y. C.; Qian, S. Y.; Tomer, K. B.; Mason, R. P. J. Biol. Chem. 2002, 277, 6104-6110. (39) Li, W.-W.; Heinze, J.; Haehnel, W. J. Am. Chem. Soc. 2005, 127, 6140-6141. (40) Hay, S.; Wallace, B. B.; Smith, T. A.; Ghiggino, K. P.; Wydrzynski, T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17675-17680. (41) Brunmark, A.; Cadenas, E. Free Radic. Biol. Med. 1989, 7, 435477.
J. Phys. Chem. B, Vol. 111, No. 13, 2007 3495 (42) Hay, S. Protein engineering of novel porphyrin/quinone-binding proteins for light-induced electron transfer. Ph.D. Thesis, The Australian National University, 2004. (43) Britz, D. J. Electroanal. Chem. 1978, 88, 309-352. (44) Berger, St.; Rieker, A. In The chemistry of the quinonoid compounds Part 1; Patai, S., Ed.; John Wiley and Sons: London, 1974; pp 163-230. (45) Santoro, M. M.; Bolen, D. W. Biochemistry 1988, 27, 8063-8068. (46) Clarke, W. M. Oxidation-reduction potentials of organic systems; Williams & Wilkins: Baltimore, MD, 1960. (47) Laviron, E. J. Electroanal. Chem. 1984, 164, 29-46. (48) Bishop, C. A.; Tong, L. K. J. J. Am. Chem. Soc. 1965, 87, 501505. (49) Zhao, J. W.; Luo, L. Q.; Yang, X. R.; Wand, E. K.; Dong, S. J. Electroanalysis 1999, 11, 1108-1111. (50) Depew, M. C.; Wan, J. K. S. In The Chemistry of the Quinonoid Compounds; Patai, S., Rappoport, Z., Eds.; Wiley: New York, 1988; Vol. 2, pp 899-962. (51) Bailey, S. I.; Ritchie, I. M.; Hewgill, F. R. J. Chem. Soc., Perkin Trans. 2 1983, 5, 645-652. (52) The surface coverage ) integrated peak area at slow scan rate/(n × F × electrode area). (53) Armstrong, F. A.; Camba, R.; Heering, H. A.; Hirst, J.; Jeuken, L. J. C.; Jones, A. K.; Le´ger, C.; McEvoy, J. P. J. Chem. Soc., Faraday Discuss. 2000, 116, 191-203. (54) Armstrong, F. A. J. Chem. Soc., Dalton Trans. 2002, 661-671. (55) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28. (56) Laviron, E.; Roullier, L. J. Electroanal. Chem. 1980, 115, 65-74. (57) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759-2766.
