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Chapter 2: the Ph-Dependent Redox Inactivation of Amicyanin As Studied by Rapid Protein- Film Voltammetry

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Chapter 2: the Ph-Dependent Redox Inactivation of Amicyanin As Studied by Rapid Protein- Film Voltammetry Chapter 2 Chapter 2: The pH-dependent redox inactivation of amicyanin as studied by rapid protein- film voltammetry Abstract The redox properties of the blue copper protein amicyanin have been studied with slow and rapid scan protein-film cyclic voltammetry. At slow scan rates, when the system is able to equilibrate with the electrode potential, the reduction potential of amicyanin depends on pH in a sigmoidal manner. The data are analysed by assuming that a single titrating group with a redox state dependent pKa is responsible for the pH-dependence of red ox the reduction potential, which gave pKa = 6.3, pKa £ 3.2 at 22 °C. Voltammetry at higher scan rates shows that the low-pH form of amicyanin is not oxidised directly. Instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion is achieved with a rate constant > 750 s-1 at 25 °C. In order to slow the coupled reaction down, the experiments were performed at 0 °C, at which a rate constant of 35 ± 20 s-1 was determined. In conjunction with evidence from NMR, the data are discussed within a scheme that incorporates protonation and dissociation of the copper coordinating histidine 96 in the reduced form. 27 Protein film voltammetry on amicyanin Introduction This paper deals with the dynamics of the pH-dependent conformational change that occurs in the active site of the small blue copper protein, amicyanin. Amicyanin functions as the obligatory electron acceptor of methylamine dehydrogenase (MADH) [Husain et al., 1985; Tobari et al., 1981; van Houwelingen et al., 1985; van Spanning et al., 1990], an enzyme that is essential when bacteria like Paracoccus versutus are grown on mimimal medium with methylamine as the sole source of carbon, nitrogen and energy [Dennison et al., 1998]. MADH is located at the beginning of the energy generating redox chain and is responsible for the conversion of methylamine into formaldehyde. As with most blue copper proteins, the single copper ion in amicyanin from P. versutus is anchored to the protein framework by two histidines (His36 and His96), a cysteine (Cys92) and a methionine (Met99) [Romero et al., 1994]. At low pH, the C-terminal histidine ligand (His96) becomes protonated and dissociates from the Cu, a property amicyanin shares with plastocyanin and pseudoazurin [Dennison et al., 1994b; Lommen et al., 1988; Sykes, 1991a]. The accompanying change from four- to three-fold coordination leads to a large increase in reduction potential [Dennison et al., 1996a], illustrating the stabilising effect the three-fold coordination has on the Cu(I) state. NMR experiments with reduced amicyanin indicate that protonation of His96 coincides also with a decrease in the self- exchange rate of amicyanin [Lommen et al., 1990]. It has been proposed that His96 might thus act as a switch that controls the physiological flow of electrons along the MADH redox chain [Guss et al., 1986; Ubbink et al., 1994]. Crystal structures of reduced plastocyanin and pseudoazurin at various pH values showed that upon protonation of the copper-coordinating histidine, the copper ion is displaced towards the plane of the remaining ligands (Sg(Cys), Nd(His) and Sd(Met)), resulting in a trigonal planar geometry [Guss et al., 1986; Vakoufari et al., 1994]. EXAFS studies on amicyanin have led to a similar conclusion [Lommen et al., 1991]. To our knowledge, protonation has never been observed when the metal is oxidised, which signifies a profound decrease of the pKa’s of these histidines. The kinetics of the dissociation/association of His96 have been studied at 25 °C by 1H NMR [Lommen et al., 1988; Lommen et al., 1990]. It appears that, when dissociated, His96 may occur in two conformations, possibly corresponding to different rotamers of the His96 side chain, similar to that observed in crystals of reduced plastocyanin at various pH 28 Chapter 2 values [Guss et al., 1986]. Since most of the mechanistic information has been obtained indirectly, it is important to complement these data by more direct measurements of redox activity. I have therefore performed cyclic voltammetry measurements on films of amicyanin immobilised on an electrode, where, by varying the pH and the scan rate, interconversion between the protonated and unprotonated forms can be observed directly. Furthermore, the variation in reduction potential can be studied over a larger range of pH values than before, leading to a more complete picture of the redox behaviour. In order to compare the results with the NMR studies, measurements were performed at 25 °C. However, since at this temperature the kinetics are very fast, the system was also studied at a second, lower temperature (0-2 °C) at which more accurate rate constants could be determined. Methods Amicyanin from P. versutus was obtained from a heterologous expression system in E. coli as described before [Kalverda et al., 1994]. For the cyclic voltammetry studies a mixed buffer system (20 mM 2-[N-cyclohexylamino]-ethanesulfonic acid (CHES), 20 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 20 mM 2-[N- morpholino]-ethanesulfonic acid (MES) and 50 mM sodium-acetate) was used. Besides the mixed buffer, the cell solutions contained 50 mM NaCl as supporting electrolyte and 2 mM neomycin sulfate was included as a co-adsorbate to stabilise the protein layer on the electrode. In the absence of neomycin, no electrochemical response was observed. The pH of the cell solutions was adjusted with HCl or NaOH at the temperature used in the experiment and checked again after the measurements. Analogue cyclic voltammetry was performed using an Autolab electrochemical analyser (Eco-chemie, Utrecht, the Netherlands) equipped with a PGSTAT 20 and a fast analogue scan generator (SCANGEN), in combination with a fast analogue to digital converter (ADC750). The electrochemical cell and pyrolytic graphite ‘edge’ (PGE) working electrode have been described previously20. Fourier transform smoothing and background subtraction were achieved using a computer programme (provided by H.A. Heering) that fits a cubic spline function [Press et al., 1989] to the baseline in regions sufficiently far from the peak and interpolates throughout the peak region. Numerical modeling was carried out as described before [Hirst et al., 1998a]. 29 Protein film voltammetry on amicyanin Protein solutions for preparing films were typically 0.5 mM in 20 mM HEPES, pH 7.0 and 2 mM neomycin. Prior to voltammetric experiments, the PGE working electrode was polished with 3 or 6 micron diamond polish (Kemet Europe B.V.) on a polishing cloth and rinsed with water. A film was applied by adding 1-2 ml of protein solution to the electrode surface with a plastic pipette-tip and retracting the solution shortly after. For NMR measurements, amicyanin (in 20 mM HEPES, pH 7.0) was reduced with sodium dithionite. The (reducing) buffer was exchanged for D2O and the sample was concentrated to ~5 mM by means of ultrafiltration. The pH was adjusted using 0.1 M NaOD or DCl, but was not corrected for the deuterium isotope effect. NMR spectra were recorded on a Bruker Avance DMX 600 MHz spectrometer and spectra were analysed using standard procedures and Bruker software. Combinations of spectra obtained with a Carr-Purcell- Meiboom-Gill (CPMG) pulse sequence [90°-t-(180°-2t)n-180°-t] (n=59; t=1ms) and a Hahn-spin-echo (HSE) pulse sequence [90°-t-180°-t] (t=60ms) were used to 0.40 22°C discriminate between singlets, doublets 0.36 and triplets in the aromatic region HE) S 0.32 [Campbell et al., 1975; Carr et al., 1954]. vs (V / 0.28 The temperature of the NMR spectrometer 0’ E 0.24 was checked by measuring the HDO shift 0.20 with respect to 3-trimethylsilylpropionate 3 4 5 6 7 8 9 0.40 (TMSP-2,2,3,3-d4). 0.36 2°C HE) S 0.32 vs (V Results and discussion / 0.28 0’ E pH-dependence of the reduction potential 0.24 Figure 1 shows the pH-dependence of the 0.20 3 4 5 6 7 8 9 reduction potential of amicyanin as pH determined with protein-film voltammetry Figure 1: Reduction potential (E0’) vs pH of at 2 °C and 22 ºC. At scan rates below 0.5 amicyanin. E0’ was measured by protein film -1 voltammetry, commencing with a reductive V s , the system is able to equilibrate with poise and with a scan rate of 0.1 V s-1. The the electrode potential (vide infra) and the voltammograms were measured at 2°C and 22°C, mixed buffer, 50 mM NaCl and 2 mM oxidative and reductive peaks of the neomycin. The line shows the least squares fit voltammograms are symmetrical at all pH using eq. 1 (see text), with values shown in Table 1. All values are referenced to the values. Note, however, that at these ‘slow’ standard hydrogen electrode (SHE). 30 Chapter 2 scan rates the peak separation (35-40 mV)* and the width at half-height (~100 mV) are greater than the ideal one-electron values (0 and 84 mV at 2 ºC, respectively and 0 and 90 mV at 22 ºC, respectively) [Armstrong et al., 1997; Hirst et al., 1998b]. Further lowering scan rates did not decrease the peak separation or width at half-height. 0’ The data were fitted to Equation 1 [Moore et al., 1990], in which E alk is the limiting value ox of the formal reduction potential at high pH when His96 is not protonated and Ka and red Ka are the proton dissociation constants of His96 in the oxidised and reduced states, respectively. æ ö K red æ K ox + éH+ ùö ç a ç a ê ú÷ ÷ 0' 0' RT ç è ë ûø ÷ E = Ealk - ln (1) nF ç ox æ red é + ùö ÷ ç Ka ç Ka + H ÷ ÷ è è ëê ûúø ø red The best fit (see Figure 1) to the data gave the values shown in Table 1.
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