ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i355 2001 © The Japan Society for Analytical Chemistry Analysis of Biological Functions of Metalloproteins Using Biocompatible Modified Electrodes Isao TANIGUCHI Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University 2-39-1, Kurokami, Kumamoto, 860-8555, Japan (E-mail: [email protected]) Recent developments on bioelectroanalytical chemistry of metalloproteins have been discussed for the following subjects. 1) Surface structures for the rapid electron transfer of metalloproteins (in particular for cytochrome c) have been discussed and 3-mercaptopyridine has been shown as a new surface modifier for cytochrome c electrochemistry. 2) Biological functions and electron transfer kinetics of myoglobin have been analyzed by comparing electrochemical properties of native molecule with those of artificially designed molecules. For electron transfer kinetics, the re-organization energy due to the structural change at the redox center during electron transfer reaction has been shown to play an important role. 3) Use of ferredoxin as an electron-donating mediator, bioelectrocatalytic reactions have been demonstrated. These results suggest that electrochemical techniques using functional electrodes are useful for analysis and use of biological functions of metalloproteins. (Received on August 10, 2001; Accepted on September 13, 2001) Electron transfer reaction is known to be one of the key 5 x 5 mm2, from Kinoene Optics Corp., Japan) for myoglobins reactions for generating a biological function: Cytochrome c is and at a surface modified In2O3 electrode for ferredoxins under an electron transfer metalloprotein in the respiratory chain. nitrogen atmosphere. Plant ferredoxin is also an important electron transfer protein Horse heart cytochrome c and myoglobin (both from in photosynthetic process. Myoglobin and hemoglobin are Sigma) were purified further by ion exchange chromatography, controlling oxygen capture and release through their oxidation as described elsewhere.2,3 Heme-reconstituted10 and mutated states. Preparation of a suitable electrode/solution interface, at myoglobin molecules were prepared and purified for which rapid electron transfer reaction of metalloprotein takes electrochemical use by chromatography. Chlorella ferredoxin place, is important not only for studying biological functions of and mutated maize11 ferredoxins were also prepared and metalloproteins using conventional electrochemical techniques purified for ferredoxin electrochemistry. 3-Mercaptopyridine but also for applying biological functions to design various and its disulfide were synthesized from 3-aminopyridine bioelectroanalytical systems. Although still many proteins according to the literature12 using improved procedures. remain unsuccessful to obtain rapid electron transfer at an electrode, in recent years various functional electrodes for Results and Discussion metalloprotein electrochemistry have been developing by many groups1 including the author's.1-4 Modified electrodes for cytochrome c electrochemistry In the present paper, recent developments on useful Cytochrome c is one of the most extensively studied functional electrodes for metalloprotein electrochemistry and proteins so far used in metalloprotein electrochemistry. bioelectrochemical systems are discussed. Indium oxide (In2O3) and so-called electron transfer promoter (such as bis(4-pyridyl)disulfide, 4,4’-PySSPy) modified Experimental electrodes are typical examples at which a well-defined voltammogram of cytochrome c is clearly seen. More recently, Au(111) single crystals were prepared by the flame- by using the atomically flat gold single crystal surfaces, annealing-quenching method developed by Clavilier et al.5 In surface structures of various modified electrodes have been situ electrochemical STM measurements of modified electrode examined at the molecular level.6, 8, 9, 13-15 surfaces were carried out in a 0.05 M HClO4 solution using a On a 4-PySH modified Au(111) electrode a well-defined Nanoscope E and an electrochemically etched tungsten tip.6 voltammetric response of cytochrome c was observed.2, 8 For The tunneling current used was around 2 nA. Surface-enhanced preparation of a suitable 4-PySH modified surface, a very IR absorption spectroscopy (SEIRAS)7 was also used to small amount of sulfide impurity in a 4-PySH-modifier evaluate the surface structure of the modified electrode. solution should be taken into account.9 The STM image for Cyclic voltammetry of cytochrome c at a single crystal the 4-PySH modified Au(111) electrode showed the electrode was carried out at 25 ˚C using the meniscus (or rectangular unit cell of a p(5 x √3R-30˚) structure with an hanging electrolyte) method.8,9 Platinum plate (1.5 x 0.7 cm) interaction of two neighboring 4-PySH.6 On the other hand, was used as a counter electrode. An Ag/AgCl (saturated KCl) the 2-PySH modified Au(111) electrode showed a p (4 x √7R- was used as a reference electrode. A BAS CV-50W 40.9˚) structure with no dimer formation, suggesting that 2- electrochemical analyzer was used for electrochemical PySH adsorbed at both thiolate S and pyridine N atoms.17 The measurements at a hydrophilic In2O3 electrode (approximately suggested structure was also confirmed by both SEIRAS and i356 ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT the electrochemical reductive desorption behavior.9, 13, 16 On a 2-PySH modified electrode, poor electrochemical response of cytochrome c was seen as was on a thiophenol modified electrode. The 2-PySH modified surface has no pyridine nitrogen at the solution side, while the 4-PySH modified surface has pyridine nitrogen faced to the solution, through which cytochrome c in solution interacts with the electrode. Similar disulfide formation for 4-PySH (p (√2R-45˚ x 5R- 53.1˚))14 and adsorption at both thiolate S and pyridine N atoms for 2-PySH ((√2 x 3√2) R-45˚) were also observed on 4- PySH and 2-PySH modified Au(100) electrodes, respectively. Again, no electrochemical response of cytochrome c was observed on a 2-PySH modified Au(100) electrode. These results suggest that for the rapid electrochemical response of cytochrome c, the pyridine nitrogen faced to solution is necessary. The above mentioned STM images of the modified electrodes were also seen not only in an acidic solution (0.05 M HClO4) but in a neutral (0.01 M NaClO4) solution. To confirm the above consideration for the suitable structure of the electrode surface, electrochemical responses of cytochrome c on 3-mercaptopyridine modified Au(111) and Au(100) electrodes were examined, because the pyridine nitrogen of 3-PySH is expected to be oriented toward the solution when 3-PySH adsorbed on gold through the thiolate- Au interaction as is observed for other mercaptopyridines. Fig. 2 High-resolution STM image of 3-PySH modified Actually, 3-PySH modified Au(111) and Au(100) electrodes Au(111) electrode at –0.05 V (vs. Ag/AgCl) in a 0.05 M showed excellent voltammetric responses for cytochrome c HClO4 solution. The (6 x √3R-30˚) structure is also shown. (Fig. 1). Clear electrochemical responses of cytochrome c were obtained in a wide pH region (3.5-9.0) on 3-PySH modified electrodes on which pyridine nitrogen faced to the The observed surface structures obtained by modifying solution. Details on the surface structure of 3-PySH modified with mercaptopyridines were the same as those obtained by Au electrodes (i.e., high-resolution STM images (Fig. 2) modifying with corresponding disulfides. suggested a (6 x √3R-30˚) structure on Au(111)) will be reported elsewhere, Probing biological functions of myoglobin (Mb) When a suitable electrode for probing metalloproteins is obtained, various biological functions related to electron- transfer reactions can be measured electrochemically. Mb is an interesting molecule, because its redox center (an iron complex of protoporphyrin-IX, protoheme-IX) is not covalently bonded to the globin moiety and is easily replaced with an artificial molecule for the redox center. Also, structure of Mb is well known on the basis of x-ray christallographic studies. Recently, binding of exogenous ligands10 and properties of semi-artificially reconstituted Mbs have been studied electrochemically.10, 18 Electron transfer kinetics was also examined by using Mbs chemically modified with cyanogen bromide.19 Electrochemical properties of various mutated Mbs were also measured at an In2O3 electrode. Roles of vinyl side-chains of protoheme-IX of Mb The redox center of native Mb has side-chains of vinyl group at 2- and 4- positions. To understand the role of vinyl groups of protoheme on Mb functions, the vinyl groups were replaced by ethyl groups or H atoms to give so-called meso- and duetero- protohemes, respectively (Fig. 3). The reconstituted Mb molecules, meso(IX)Mb and deutero(IX)Mb prepared by using these redox centers, showed well-defined redox waves at an In2O3 electrode in a 0.1 M bis-Tris buffer solution (pH 6.5). The E0’ values obtained were –140 mV for Fig. 1 Cyclic voltammograms of 0.1 mM cytochrome c (Cyt. native Mb, –162 mV for deutero(IX)Mb and –170 mV for c) in a phosphate buffer solution (pH 7) at 2-, 3- and 4-PySH meso(IX)Mb, indicating that native Mb is more easily reduced modified Au(111) electrodes at a scan rate of 50 mV/s, than others. Since Mb is in reduced form for oxygen storage in together with schematic illustrations of surface structures. biological systems, the vinyl groups of heme would keep Mb easy to be reduced. ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i357 Mb. To make sure this consideration, some mutated myoglobin molecules, of which structures are known from the Brookheaven Protein Data Bank, were used as shown in Fig. 4. H64V mutated Mb (from pig heart), for which no water molecule is coordinated to the heme iron, gave the k0’ value of 2.5 (± 0.5) x 10–3 cm s-1 (E0’ = –84 mV vs. Ag/AgCl), while native pig heart Mb showed the k0’ value of 3.4 (± 0.5) x 10–4 cm s–1 (E0’ = –133 mV vs.