Article pubs.acs.org/JPCC
Electrochemical Behavior of Cytochrome c552 from a Psychrophilic Microorganism Olga M. Sokolovskaya, John S. Magyar, and Marisa C. Buzzeo* Department of Chemistry, Barnard College, Columbia University, 3009 Broadway, New York, New York 10027, United States S Supporting Information *
ABSTRACT: Psychrophilic organisms play a significant role in global nutrient cycling and bioremediation, and thus there is great interest in understanding the details of their cellular processes. Here, we report the first electrochemical measurements of cytochrome c 552 isolated from the model psychrophile, Colwellia psychrerythraea. Quasi-reversible redox behavior is observed at thiol-modified and bare gold electrodes with midpoint potentials of 247 and 277 mV vs SHE, respectively. Comparison of the voltammetric response on the two substrates illustrates the need for a modified surface to direct protein orientation and facilitate electron transfer in a reproducible manner. Temperature-dependence measurements of the current profile illustrate the protein’s ability to participate in electron transfer reactions over a 50° range, which is supported by recent structural evidence of enhanced flexibility. These electrochemical findings allow the behavior of this psychrophilic cytochrome to be evaluated in the context of meso- and thermophilic electron transfer proteins.
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INTRODUCTION Psychrophilic organisms are known to participate in global nutrient cycling and bioremediation, yet little is understood about how they operate so efficiently at low temperatures.1−8 While their proteins do not differ significantly in structure from their mesophilic9−12 or thermophilic counterparts,13−17 analysis of the amino acid composition reveals lower numbers of proline and arginine and higher numbers of glycine, serine, and methionine.2 Notable trends also include fewer hydrogen bonds and ionic interactions, reduced hydrophobicity in internal residues, and increased solvent interactions through apolar outer residues.18−21 Increased flexibility, either locally or globally, is now considered central to cold adaptation. Colwellia psychrerythraea, a psychrophilic marine bacterium isolated from Arctic and Antarctic sediments, is an ideal model psychrophile for studies on bacterial cold adaptation. This microorganism requires temperatures from −1 to 10 °C to grow, and its full genome sequence was published in 2005.22 Protein folding dynamics and electron transfer studies of this system will contribute to the overall understanding of psychrophiles’ function, flexibility, and adaptability. The molecular structure of the electron transfer protein cytochrome c552 isolated from Colwellia psychrerythraea (Cpcyt c552) was recently published.23 On the basis of structural comparisons of psychro- and mesophilic homologues, a methionine-based ligand-substitution mechanism for the stabilization of Cpcyt c552 was proposed. We are interested in examining the voltammetric behavior of Cpcyt c552 in light of these structural features to better understand the protein’s ability to partake efficiently in electron transfer reactions at such low temperatures. © 2014 American Chemical Society
Several reports have demonstrated the applicability of voltammetric measurements to the characterization of electron transfer proteins24−33 and specifically to the study of cytochromes.34−60 Careful consideration must be given to electrode preparation, substrate modification, and spatial arrangement of these bulky, charged molecules on the conducting surface. Electrostatic interactions between exposed amino acids and charged surface modifiers can encourage preferential and reproducible protein orientation and thus establish a reliable and efficient pathway for electronic communication between the redox-active metal center and the electrode. Electrochemical studies of Cpcyt c552 will afford evaluation of the psychrophile’s intrinsic redox activity relative to the behavior of its meso- and thermophilic analogues. Temperature-dependence studies will allow for the stability and flexibility of the electron transfer protein to be assessed in the context of probable structural accommodations. We ultimately seek to identify the molecular characteristics that make these psychrophilic organisms suitable to function at low temperatures so as to better understand their role in biogeochemical cycling and bioremediation.
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EXPERIMENTAL PROCEDURES General/Electrochemistry. All voltammograms were recorded on an Autolab PGSTAT20 potentiostat (Metrohm USA, Riverview, FL) using commercially available gold Received: January 31, 2014 Revised: July 28, 2014 Published: July 29, 2014 18829
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Direct Protein Electrochemistry. Solutions of Cpcyt c552 were prepared by redissolving lyophilized purified protein into chilled phosphate buffer (50 mM PBS, pH 7). The concentration of protein used in these experiments ranged from ∼20 to 200 μM.
macroelectrodes (1.6 mm diameter; BASi, West Lafayette, IN). Platinum wire (0.5 mm diameter; Alfa Aesar, Ward Hill, MA) and Ag/AgCl (BASi) electrodes were used as the counter and reference, respectively. A water-jacketed electrochemical cell and a peristaltic pump (Watson-Marlow, Inc., Wilmington, MA) allowed the temperature of the electrode surface and analyte solution to be controlled. Cyclic voltammograms were recorded at a scan rate of 50 mV/s, unless otherwise noted. The following parameters were used for square wave voltammograms: step potential, 5 mV; amplitude, 20 mV; and frequency, 25 Hz. Potentials are reported either versus standard hydrogen electrode (SHE) or Ag/AgCl. All solutions were degassed with ultrahigh-purity nitrogen, and a blanket of nitrogen was maintained above the solution throughout the measurements to ensure an inert atmosphere. Chemicals. Ethanol, hexanethiol (HXT), 6-mercaptohexanoic acid (MCHA), 8-mercapto-1-octanol (MCO), 8mercaptooctanoic acid (MCOA), octanethiol (OCT), potassium chloride, sodium chloride, sodium monobasic phosphate, and sodium dibasic phosphate were all purchased from SigmaAldrich. Potassium ferricyanide was purchased from Acros Organics. Ultrahigh-purity nitrogen was purchased from Airgas. All chemicals were used as received. Protein Isolation and Purification. As described previously by Magyar and co-workers,61 a synthetic gene (GeneWiz, Inc., South Plainfield, NJ) for Cpcyt c552 was coexpressed in E. coli with the pEC86 heme cassette developed by L. Thöny-Meyer and co-workers.61,62 The resulting protein includes the expected covalently bound heme group and was purified to homogeneity by cation exchange chromatography.61 Purified protein was characterized by mass spectrometry (calculated m/z = 8832; experimental m/z = 8834) and UV− vis spectroscopy (Figure S1, Supporting Information). Electrode Modification. Gold surfaces were prepared by mechanically polishing rod electrodes with alumina suspensions of decreasing particle size (1.0, 0.3, and 0.05 μm) on TexMet and Microcloth polishing pads (Buehler, Lake Bluff, IL). Freshly polished electrodes were submerged into Eppendorf tubes containing thiol solutions and covered with Parafilm to prevent evaporation. The following ethanolic solutions were used for surface modification: 200 μM 1:1 MCOA/OCT, 300 μM 2:1 MCOA/OCT, 200 μM MCOA, 300 μM 2:1 MCHA/ HXT, 200 μM 1:1 MCOA/HXT, and 300 μM 2:1 MCOA/ HXT. Electrodes were incubated in thiol solutions at 4 °C for a minimum of 3 h. Following incubation, electrodes were rinsed thoroughly with ethanol, deionized water, and phosphate buffer (10 mM PBS, pH 7) prior to electrochemical measurement. Monolayer formation was monitored by cyclic voltammetry in a deoxygenated solution of 0.2 mM K3[Fe(CN)6] in 0.1 M KCl/ H2O. Protein Immobilization. Purified Cpcyt c552 was resuspended in chilled deionized water to a concentration of at least 300 μM. The concentration was estimated by measuring the absorbance of the solution at 523 nm using a Nanodrop spectrophotometer (l = 0.1 cm, ε ∼ 9,500 M−1cm−1).61 Modified electrodes were plated with ∼35 μL of cytochrome c solution immediately after rinsing off excess thiol and were then incubated overnight at 4 °C in a humidifier chamber to prevent evaporation. Electrodes were rinsed with chilled phosphate buffer (10 mM PBS, pH 7) prior to voltammetric measurement. Surface coverage was estimated to be ∼3 × 10−11 mol/ cm2 at 6 °C.
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RESULTS AND DISCUSSION Prior to electrochemical measurement of Cpcyt c552, experimental conditions were optimized to ensure reproducible surface coverage by alkanethiol modifiers. Modification of the gold electrodes was evaluated by measuring the extent of passivation against solution-borne ferricyanide by cyclic voltammetry. Candidate modifiers included hexanethiol (HXT), 6-mercaptohexanoic acid (MCHA), 8-mercapto-1octanol (MCO), 8-mercaptooctanoic acid (MCOA), and octanethiol (OCT). Composition, concentration, and incubation time were adjusted and passivation results compared to identify the optimal conditions (Table S1, Supporting Information). Both the ratio of neutral to charged thiol modifiers and the alkane chain length were varied to identify a surface that would best promote immobilization of the protein via electrostatic interactions. Of these monolayers, mixed compositions of MCOA with either OCT or HXT hindered diffusion of ferricyanide to the electrode most effectively and reproducibly. A typical voltammetric response for a gold surface modified with a mixed monolayer of 1:1 MCOA/OCT in the presence of 0.2 mM ferricyanide is illustrated in Figure 1. Excellent passivation is observed in the potential window of interest.
Figure 1. Cyclic voltammogram (CV) of degassed 0.2 mM K3[Fe(CN)6]/0.1 M KCl/H2O on a gold electrode incubated with 1:1 MCOA/OCT. Inset: CV of 0.2 mM K3[Fe(CN)6]/0.1 M KCl/ H2O on bare gold. Scan rate: 100 mV/s.
Following optimization of monolayer formation conditions, Cpcyt c552 was immobilized on modified gold electrodes for electrochemical studies. The electrodes were first incubated with alkanethiol solutions, rinsed thoroughly, and subsequently incubated with protein solution overnight. Figure 2 shows the stable, quasi-reversible voltammetric response observed for the reduction and oxidation of Cpcyt c552 on electrodes modified with 2:1 MCOA/OCT. Similar results were recorded on electrodes incubated with 2:1 MCOA/HXT and 1:1 MCOA/ HXT. The carboxylic acid head groups of the MCOA modifier are believed to interact favorably with positively charged lysine residues on the face of the cytochrome nearest to the central 18830
dx.doi.org/10.1021/jp501146e | J. Phys. Chem. C 2014, 118, 18829−18835
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Figure 2. CV of Colwellia psychrerythraea cytochrome c552 (Cpcyt c552) in degassed phosphate buffer (10 mM PBS, pH 7) at 6 °C on a gold electrode incubated with 2:1 MCOA/OCT. Inset: CV in degassed phosphate buffer solution prior to protein incubation.
Figure 3. Square wave voltammograms (SWV) for Cpcyt c552 in degassed phosphate buffer (10 mM PBS, pH 7) on a gold electrode modified with 2:1 MCOA/HXT. Temperatures recorded: 8, 21, 32, 43, 50, and >55 °C.
heme group.43,45−50,53−59,63 This preferential association allows for more reproducible alignment of the bulky protein atop the monolayer and therefore provides a consistent electron transfer pathway. A mixture of alkane chain lengths and terminal functional groups within the same monolayer also proved favorable for protein orientation. Previous reports have highlighted the enhanced electron transfer observed on carboxylate-terminated alkanethiol monolayers diluted with shorter methyl- or hydroxyl-terminated chains.35,45,49,54 The heterogeneous composition is thought to provide an optimal surface with which the protein molecules can interact while retaining their native reactivity. Interestingly, the observed midpoint potential of 247 mV vs SHE (50 mV vs Ag/AgCl) is remarkably close to values reported for meso- and thermophilic cytochromes on gold substrates.44−46,48−52,54,60,63,64 Given the striking differences in these organisms’ growth conditions and natural environments, the similarity in reduction potentials is of particular note and suggests that Cpcyt c552 interacts with similar electron transfer partners as its mesophilic counterparts (e.g., cytochrome c peroxidase and nitrous oxide reductase). The above electrochemical measurements were recorded at low temperatures (4−8 °C) to mimic the psychrophile’s native setting. To probe the thermal stability of the protein’s redox activity, we next examined the voltammetric response of Cpcyt c552 over a 60° range. Measurements were first recorded at 5 °C, and then the temperature was increased in 5° to 10° increments. As is illustrated by the square wave voltammograms in Figure 3, the current response clearly diminishes as the temperature is raised. By a temperature of 35 °C, voltammetric peaks are difficult to distinguish. Two important controls were conducted to ensure the observed dependence was a reflection of the protein’s activity. First, we examined the stability of the thiol monolayer (in the absence of protein) as a function of temperature. Modified electrodes were tested for passivation against potassium ferricyanide and then placed into a solution of phosphate buffer and heated to about 40 °C. Passivation was retested, and no significant difference was observed, suggesting that the monolayer is not irreversibly altered during heating. Second, the stability of the current response was evaluated as a function of the time the modified electrode was submerged in solution. A decrease in current was seen during the first 10 min, but the signal quickly equilibrated and remained visible after an hour. Given this observation, all temperature measurements
were recorded once the electrode had been immersed in the cell for 10 min. The temperature dependence illustrated in Figure 3 is therefore attributed to a change in the protein’s behavior at the modified gold surface. The current decrease could be the result of several different surface events, including, though not limited to, protein unfolding (reversibly or irreversibly), protein reorientation, and loss of immobilized protein. To examine the surface behavior further, the extent to which the redox signal could be restored, following exposure to elevated temperatures, was measured. Indeed, the reappearance of peaks is observed upon cooling of an electrode which had been heated to temperatures above 50 °C. Figure 4 shows an overlay
Figure 4. SWV for Cpcyt c552 in degassed phosphate buffer (10 mM PBS, pH 7) on a gold electrode modified with 1:1 MCOA/HXT at 4 °C (squares), 23 °C (triangles), and again at 4 °C (circles).
of three square wave voltammograms of Cpcyt c552 recorded on the same modified surface (1:1 MCOA/HXT) at 4 °C, at 23 °C, and again at 4 °C. Upon heating the electrochemical cell, a loss in voltammetric signal is observed relative to the original scan. The current is partially restored, however, when the solution is cooled back below 10 °C. This same pattern was observed for protein solutions heated to 30 °C, to 49 °C, and to 55 °C. Signal restoration was not reliably observed after raising the temperature to 65 °C. Preliminary analysis of the potential dependence on temperature yields thermodynamic 18831
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estimates for the electron transfer reaction (ΔH° = −42.5 kJ mol−1; ΔS° = −70.3 J mol−1 K−1) that agree reasonably well with corresponding values reported for meso- and thermophilic cytochrome c systems.60,65−69 The observed voltammetric results suggest that above 10 °C the optimal electron transfer pathway is compromised (yet still functional). The increase in temperature likely encourages the protein to access different orientations of varying redox activity, which will be reflected in the current response. In certain conformations and at certain temperatures, the distance from the central heme to the electrode surface will be lengthened, thus attenuating electron transfer. Importantly, however, the protein appears to remain associated with the charged monolayer even at elevated temperatures and is able to participate again fully in redox chemistry once cooled. This electrochemical behavior is consistent with recent spectroscopic and structural studies of Cpcyt c552 by Magyar and co-workers. Monitoring of protein folding by UV−vis absorption spectroscopy revealed an unfolding midpoint temperature of 61 °C, thus demonstrating the remarkable thermal stability of this psychrophilic protein.61 Comparison of the cytochrome’s molecular structure to mesophilic counterparts suggests the protein’s stabilization is achieved via a ligand-substitution mechanism, in which neighboring methionine residues are able to misligate the heme upon loss of the native ligand.23 This built-in flexibility, made plausible by an abundance of methionine residues in the heme’s vicinity, increases the protein’s stability and decreases the likelihood of irreversible denaturation. Further temperature-dependence spectroelectrochemical studies of Cpcyt c552 will allow correlations to be drawn between the thermodynamics of the electron transfer reaction and the structural differences in the heme’s environment. For comparison, the electrochemical response of Cpcyt c552 was also tested on bare gold surfaces. As seen in Figure 5, quasi-
currents showed a linear dependence on the square root of the scan rate, indicative of a diffusional controlled response (Figure 5, inset), and the measured peak currents were on the same order of magnitude as those predicted using the diffusion coefficient for horse heart cytochrome c.47 The voltammetric response scaled linearly with increasing protein concentration with distinct peaks observed at concentrations as low as ∼20 μM. Not surprisingly, the temperature dependence of Cpcyt c552 on bare gold differed significantly from the response at alkanethiolate surfaces. In Figure 6, it can be seen that the
Figure 6. CV of Cpcyt c552 (∼100 μM) on a bare gold electrode in degassed phosphate buffer (50 mM PBS, pH 7) with increasing temperature: 6 °C (solid line), 22 °C (dashed line), and 38 °C (dotdashed line). Inset: CV of Cpcyt c552 on the same bare gold electrode after the temperature is raised above 65 °C.
voltammetric signal initially increases as a function of temperature, as there is faster diffusion of the solution-borne cytochrome to and from the electrode surface. The redox peaks eventually begin to decrease above 40 °C, however, as both structural changes and surface adsorption compete with the enhanced diffusion; voltammetric activity is lost entirely above 65 °C (Figure 6, inset). Consistent with previous reports, passivation of bare gold by nonspecifically adsorbed protein proved to be a substantial limitation of this approach.70−72 Particularly at elevated temperatures, the electrodes had to be polished repeatedly to maintain a signal, making it impossible to distinguish between temperature-induced and surface effects. These findings underscore the need for a charged and directing surface structure to achieve reproducible electrochemical measurements of cytochromes on gold substrates.
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CONCLUSIONS Electrochemical characterization of Colwellia psychrerythraea cytochrome c552 (Cpcyt c552) on modified and bare gold electrodes reveals quasi-reversible voltammetry with peaks centered at 247 and 277 mV vs SHE, respectively. The midpoint potential of this psychrophilic protein is notably similar to its meso- and thermophilic analogues. Modification of gold surfaces with a charged and mixed monolayer is required to obtain reproducible protein voltammetry that is not compromised by nonspecific protein adsorption. Measurements on alkanethiolate surfaces illustrate the electron transfer protein’s ability to function over a 50° range, despite its native growth conditions of
