Electrochemical, resonance Raman, and surface enhanced Raman spectroscopic study of biomolecules: cytochrome c and its mutants

Rat wild-type cytochrome c (WT) and its mutants, Tyr67Phe, Thr78Val, AsnS2Ile, AspSOArg, and His26Val were studied by cyclic voltammetiy (CV), resonance Raman (RR), and surface-enhanced resonance Raman scattering (SERRS) spectroscopy. Both SERRS and CV results show that most of the cytochrome c mutants behave similarly to the wild-type cytochrome c at electrodes in tenn of reversibility except for the His26Val mutant However, the so-called "water mutants" Tyr67Hie, Thr78Val, and AsnS2ne have much lower redox potentials than that of the wild-type cytochrome c which indicates that these mutations increase the stability of the protein. The resonance Raman spectra of the water mutants also show considerable differences firom the wild-type, especially in the low frequency region which indicates structural changes in the heme/protein interactions. The surface-enhanced resonance Raman results indicate that the heme group of the adsorbed cytochrome c and its mutants are in the six-coordinate low spin (6cLS) configuration at bis(4-pyridyl) disulfide-modifled-silver electrodes, whereas mixtures of both 6cLS and nve-coordinate high spin (5cHS) heme configurations are present at bare silver electrodes. The structural basis for these results are discussed. 87 Introduction Protein electiochemistiy is a subject of great interest and has been extensively developed in recent years [1-9] due to the fact that there are some analogies between the electrode reaction of proteins and their biological electron transfer reaction. Such studies can yield important information not only about themfiodynamic, kinetic and structural properties of proteins, but also provide novel insights into the electron transfer mechanism of proteins in vivo. Mitochondrial cytochrome c is one of the most extensively studied proteins because it plays important role in energy transduction and it is relatively stable under in vitro conditions. Its structural and chemical reactivity are well-characterized, and it provides a paradigm for other globular proteins [10]. Many studies have been focused on developing and understanding the structure/function relationships in redox proteins [2,4]. Evidence as to the role of specific amino acids has been gleaned from correlation of extensive data obtained from high resolution crystallography, 2-D-Nosey nmr spectroscopy and amino acid analysis of eukaryotic cytochromes [11,12]. Extensive studies of cytochromes labeled with redox active species have established that the intramolecular electron transfer kinetics are affected by the amino acid pathway [13,14]. It is also well- known that the redox potential of the heme is influenced strongly by the nature of the axial ligand to iron in heme-containing proteins, as well as the hydrophobicity of the heme environment [15,16]. Recently, site-directed mutagenesis of invariant amino acids near the heme, but not directly ligated to it, have established that these substitutions can have considerable effect on the protein stability [10]. Resonance Raman spectroscopy is a useful technique for characterization of the 88 structural features of proteins. It can be used to identify the presence or absence of particular structural and ligation interactions of the amino acids [17]. Therefore, it is veiy powerful method for correlating structure and fiuiction. It is also an extremely sensitive technique for selectively probing the heme moiety of cytochromes. Vibrational modes have been assigned that are sensitive to the oxidation and spin state of the heme iron, as well as to distortions in the heme imposed by the surrounding protein [18]. Surface- enhanced resonance Raman spectroscopy provides addition information conceming the stability, redox potential, and configuration of the protein on an electrode surface [19]. In the present study, several cytochrome c mutants have been characterized by cyclic voltammetry, RR and SERRS in order to determine how the mutations affect the redox potential, electron transfer kinetics and the stability of cytochrome c. Experimental The procedures for preparing the mutant cytochromes have been described previously [10]. Bis(4-pyridyl) disulfide (4-PyS)>98% purity was purchased from Aldrich Chemical Co. and was used as received. All other chemicals were reagent grade without further purification. The electrochemical measurements were carried out in a conventional three-electrode electrochemical cell using a BAS 100 electrochemical analyzer (Bioanalytical Systems). The working electrode were constructed from a flattened gold or silver rod which was sealed into glass tube using Torr Seal (Varian Co.). A Pt wire was used as the auxiliary electrode. A saturated calomel electrode (SCE) served as a reference electrode and all the potentials are reported with respect to the SCE. The working electrodes were sequentially polished with 5.0, 0.3, 0.05 pm alumina slurries in 89 water. The electrodes were sonicated in distilled water following each step of the polishing procedure. Modification of the surface was achieved by dipping the electrode into a 1 mM solution of 4-PyS for 10 minutes, followed by rinsing with distilled water and immersion into the electrochemical cell containing KT* M cytochrome c solution, 10 mM potassium phosphate buffer (pH 7) and O.S M KCl for subsequent electrochemical measurements. For the SERRS measurements, after polishing, the silver electrode was roughened in 0.1 NajSQf by stepping the potential firom -O.SS V to -tO.S5 V, where 2SmC/cm^ charge was allowed to pass, and then stepping back to -0.SS V to reduce the Ag^. The electrode was then nnodified by the procedure described above for the electrochemical measurements. The SERRS studies at modified silver electrodes were accomplished by adsorbing the cytochrome c from 0.1 mM solution for 15 minutes, rinsing the excess solution from the electrode with buffer and immersing the electrode into 10 mM potassium phosphate buffer 7) containing O.S M KCl as the electrolyte. The RR and SERRS spectra were obtained by excitation with the 413 nm line of a Kr* laser. A backscattering geometry was used for collecting the scattered light The Raman instramentation has been described previously [20], This includes a Triplemate 1377 spectrometer and a Princeton Instrument CCD detector (model LN1152) cooled to -120°. Typically, the RR and SERRS data were acquired with ca. 1 mW of power and a total acquisition time of less than 2 minutes. Indene was used to calibrate the Raman frequencies. 90 Results and Discussion The molecular structure of cytochrome c is illustrated in Hgure 1 [21]. The mutants examined in this study are marked in Figure 1 including the following: Tyrosine 67 taken to phenylalanine (Tyi67Phe), asparagine 52 taken to isoleucine (AsnS2ne), threonine 78 taken to valine (Thr78Val), aspartate SO taken to aipnine (AspSOArg), and histidine 26 taken to valine (His26Val). Figure 2 shows the CV response for cytochrome c Tyr67Phe mutant at a 4-PyS-modified gold electrode at different scan rates. A plot of the peak current for both the cathodic and anodic peaks shows a linear increase as a function of the square root of die scan rate. The same behavior was observed for wild- type cytochrome c and all of the other mutants, demonstrating that the electrochemical reaction at 4-PyS-modified gold electrode is diffusion-controlled. The electrochemical results are sunrunarized in Table 1. From die peak separations of Table 1 it can be seen that most of the cytochrome c mutants have similar electrochemical kinetics to the wildtype cytochrome c, except His26Val mutant This is not suiprising, since aspartic acid (Asp) 50, tyrosine (Tyr) 67, threonine (Thr) 78 and asparagine (Asn) 52 are internal amino acids and their substitution by other amino acids does not significantiy change the protein surface structure and, hence, its interaction with the electrode. These results agree well with the hypothesis [2,4] that the promoter modified electrode surface does not recognize cytochrome c molecule very rigorously. The surface lysine residues around the exposed heme edge of cytochrome c, such as Lys 27 and Lys 72, have been suggested to be involved in binding to an electrode surface [22]. Since an electrode covered with a monolayer of bis(4-pyridyl) disulfide is an ideally hydrophilic surface, substitution of the