Accepted Manuscript Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs

David Novak, Milos Mojovic, Aleksandra Pavicevic, Martina Zatloukalova, Lenka Hernychova, Martin Bartosik, Jan Vacek PII: DOI: Reference:

S1567-5394(17)30364-X doi:10.1016/j.bioelechem.2017.09.011 BIOJEC 7056

To appear in:

Bioelectrochemistry

Received date: Revised date: Accepted date:

18 July 2017 19 September 2017 19 September 2017

Please cite this article as: David Novak, Milos Mojovic, Aleksandra Pavicevic, Martina Zatloukalova, Lenka Hernychova, Martin Bartosik, Jan Vacek , Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biojec(2017), doi:10.1016/j.bioelechem.2017.09.011

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ACCEPTED MANUSCRIPT

Electrochemistry and Electron Paramagnetic Resonance Spectroscopy of Cytochrome c and its

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Heme-Disrupted Analogs

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David Novak,a Milos Mojovic,b Aleksandra Pavicevic,b Martina Zatloukalova,a,c Lenka

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Hernychova,d Martin Bartosikd and Jan Vaceka,*

a

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry,

Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade,

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b

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Palacky University, Hnevotinska 3, Olomouc 775 15, Czech Republic

c

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Serbia

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Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw,

d

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Zwirki i Wigury 101, 02-089 Warsaw, Poland

Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute,

Zluty kopec 7, Brno 656 53, Czech Republic

*

) corresponding author: Department of Medical Chemistry and Biochemistry, Faculty of

Medicine and Dentistry, Palacky University, Hnevotinska 3, Olomouc 775 15, Czech Republic; E-mail: [email protected]; Tel.: +420-585-632-303; Fax: +420-585-632-302

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT Abstract Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electrontransfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin

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cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and

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spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of

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this report is a complex evaluation of cyt c reduction and oxidation at the level of surfacelocalized amino acid residues and the heme moiety in a single electrochemical scan. The

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electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c,

cytochromes and other hemeproteins.

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Keywords

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which could be applicable in further studies on the redox properties and structural stability of

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type-c cytochrome, chronopotentiometry and voltammetry, electron paramagnetic resonance,

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heme, hemin, hemoproteins

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT 1. Introduction Cytochrome c (cyt c) is a conjugated, highly conserved protein with a molecular weight of ~12 kDa, composed of a basic polypeptide chain and a non-protein prosthetic group heme. Being an essential component of the respiratory chain as a mitochondrial electron transporter, it affects many regulatory processes involved in homeostasis or apoptosis, as well as other

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regulatory processes occurring in mitochondria [1, 2]. There is a broad spectrum of methods

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available for the physico-chemical and structural characterization of around 300 different

spectroscopic

techniques

[4],

high-resolution

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types of cyt c [3]. The main interest is oriented towards development of new fluorescence nuclear

magnetic

resonance

[5],

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immunochemical biosensing approaches [6] and molecular dynamics calculations [7]. Due to the presence of non-protein redox-active heme moieties, electrochemical

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techniques, such as direct voltammetry (or protein film voltammetry) have been developed for investigation of cytochromes [8]. The original approach developed by Eddowes and Hill [9] further

improved

by

many

authors,

including

the

recent

application

of

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was

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spectroelectrochemical methods, which enable the monitoring of both the redox and optical activity of the heme component [10]. Moreover, great progress was made in using carbon,

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gold, metal oxide, nanostructured or self-assembled monolayer-modified electrodes in the analysis of cyt c redox activity, as reviewed in [11].

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In this work, we report a novel strategy suitable for the electroanalysis of cyt c. We show that in a single chronopotentiometric or voltammetric scan, it is possible to monitor response of both the prosthetic group and polypeptide component of the protein. The proposed method is based on an application of cathodic chronopotentiometry [12] and anodic voltammetry [13], previously applied in the electrochemical sensing of structural changes, chemical modifications and intermolecular interactions of non-conjugated proteins and glycoproteins (reviewed in [14]). We monitored both the catalytic and non-catalytic

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT electrochemical responses of adsorbed cyt c and its structural analogs, apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c) [15], which are the proteins where the heme and Fe atom moiety are eliminated, respectively. The electrochemical data are complemented and rationalized by low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy, extending previously reported observations [16, 17]. Interpretation of

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experimental data was performed using 3D visualization of redox centers based on molecular

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models obtained from high-resolution X-ray diffraction data [18].

2. Experimental

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2.1. Chemicals and General Methods

Cyt c isolated from bovine or horse heart and hemin were purchased from Sigma-Aldrich (St.

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Louis, MO, USA). Cyt c analogs acyt c and pcyt c were prepared according to modified protocol [15]. Isolation of acyt c and pcyt c monomers was performed using Sephadex G-25

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and Sephacryl S100HR (Sigma-Aldrich), respectively. The dialysis with Novagen D-tube 3.5

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kDa (Merck, USA) was used as the final purification step. Cyt c mouse monoclonal antibody (A-8), rabbit polyclonal antibody (H-104) and secondary antibodies, goat anti-rabbit IgG HRP

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and goat anti-mouse IgG HRP, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals of analytical grade were obtained from Sigma-Aldrich or Merck

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(Darmstadt, GE). The pH measurements were carried out with a HI 2211 pH/ORP Meter (HANNA instruments, IT). Protein concentration was strictly adjusted by the means of two independent spectrophotometric techniques [19, 20].

2.2. Electrochemistry Cyt c samples were analyzed by square-wave voltammetry (SWV), a.c. voltammetry (ACV) and constant-current chronopotentiometric stripping (CPS) analysis using an adsorptive pre-

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT concentration step [21]. SW voltammograms are expressed as current I(net) vs. potential E. ACV was performed in phase-out and phase-in modes according to previously reported protocol [22]. In CPS measurement, constant-current density is applied to the working electrode and the resulting variation of the interfacial potential is measured in time. The resulting raw (E–t) curves are plotted as derived parameter (dE/dt)-1 vs. E [14, 23].

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Two types of working electrodes were used: HMDE (hanging mercury drop electrode;

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area 0.4 mm2) and a basal-plane pyrolytic graphite working electrode, PGE (9 mm2, source of PG: Momentive Performance Materials, USA). Electrochemical analyses were performed

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with a μAutolab III analyzer (Metrohm Autolab, NL) in a three-electrode setup with a

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Ag/AgCl/3M KCl electrode as a reference and a platinum wire as the auxiliary electrode. For PGE surface renewal/pretreatment we used a procedure based on applying potential +1.7 V

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for 60 s in the supporting electrolyte and peeling-off the graphite top layers using sticky tape [21]. All experiments were done at 25 ºC in the presence of air.

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Dithiothreitol (DTT)-modified HMDE was prepared after the immersion of the bare

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electrode into 0.1 M Na-phosphate buffer (pH 7) containing 1 mM DTT. The DTT layer formation was performed at –0.1 V for 60 s according to previously reported protocol [24,

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25].

The parameters for both electrochemical methods used were as follows. CPSA

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parameters: accumulation time 10 s, initial (0 V) and end (–1.95 V) potential, stripping current –12.5 µA. SWV parameters: accumulation time 10 s, initial (0.2 V) and end (+1.25 V) potential, step potential 5 mV, amplitude 25 mV and frequency 200 Hz.

2.3. EPR Spectroscopy All EPR measurements were performed using an ELEXSYS II E-540 EPR X-band spectrometer.

Electroactivity of Cytochrome c and its Structural Analogues

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2.3.1. Low-temperature Measurement Cyt c samples were dissolved in 0.1 M Na-phosphate buffer at pH 7 for low-temperature measurements. The samples were transferred to the Suprasil CFQ cuvettes and placed into the EPR cavity equipped with a liquid helium cryostat. The recording parameters were:

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temperature 19 K, microwave frequency 9.38 GHz, sweep width 3500 G, microwave power

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6.325 mW, modulation amplitude 5 G and modulation frequency 100 kHz.

2.3.2. Spin-probe Labeling

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For 3-maleimido-proxyl (5-MSL) labeling, cytochromes were dissolved in 0.1 M Naphosphate buffer (pH 7). 5 μl of 5-MSL dissolved in ethanol was added at tenfold molar

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excess to 500 µl aliquots of the working protein solutions, and the samples were incubated for 24 h at 4 °C. The unbound label was removed by dialysis; see section 2.1. Afterwards, the

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samples were withdrawn into teflon tubes (Zeus Industries Inc.) and placed into the EPR

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cavity. The recording parameters were: microwave frequency 9.85 GHz, sweep width 200 G,

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microwave power 10 mW, modulation amplitude 2 G and modulation frequency 100 kHz.

2.4. Other Complementary Methods

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Electrophoresis and Western blotting were carried out as reported [26]. For UV-Vis spectroscopy experiments a UV-2401 PC spectrophotometer (Shimadzu, JP) was used. Mass spectrometry data were collected using an Orbitrap Elite (Thermo Fisher Scientific, USA) instrument connected to an UltiMate 3000 RSLCnano chromatograph (Thermo Fisher Scientific, USA) in accordance with published results [27]. The 3D molecular models were prepared in PyMOL (Molecular Graphics System, v1.7.4.5 Schrodinger, LLC) where the X-

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT ray-determined high-resolution structure of cyt c (bovine) was taken directly from the Protein Data Bank (2B4Z) [18].

2.5. Data Evaluation Data were collected using GPES ver. 4.9 (Metrohm Autolab, NL). For data post-acquisition

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processing eL-Chem Viewer ver. 1.0 [28] and Microsoft Excel 2003 Professional Edition

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were used. CPS records were processed using smooth level 3 in GPES software. This

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operation is the reason for small artificial kink, which can be observed prior shoulders of CPS peaks.

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The relative values of ± S.D. for area of electrochemical peaks (n=6) were as follows: peak H (6.1 %), peak HM (6.8 %), peak XY (11.5 %) and peak HM’ (13.5 %). These S.D.

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values are based on independent (de novo) preparation of cyt c samples in purified monomeric form prior each analysis. S.D. values for peak potentials varied in the scale ± 6 mV (n=12) for

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both CPSA and SWV.

3. Results and Discussion

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3.1. Sample Preparation and Validity Evaluation We studied cyt c and its two structural analogs, acyt c and pcyt c. Both disrupted cytochromes

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were isolated from bovine and horse heart and purified as reported [15, 29]. Acyt c was prepared from native cyt c by precipitating the heme component using Ag2SO4 in acidic medium, followed by the protein reduction in presence of dithiotreitol (DTT) and guanidine hydrochloride. Pcyt c was obtained by selectively removing the heme iron using anhydrous hydrofluoric acid. Protein samples were first validated with UV-Vis spectrophotometry, showing typical absorption patterns for all three structural forms (Fig. 1A), which were in agreement with

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT literature data [15, 29, 30]. Observed changes in absorbance of acyt c as compared to the native protein and pcyt c can be ascribed to heme elimination. The integrity of the samples was further validated using SDS-PAGE and Western blotting, with a resulting purity of ≥ 95 %. The affinity of both A-8 and H-104 antibodies towards all cyt c samples was immunologically verified, which can be useful for further methodological studies (shown for

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H-104 in Fig. 1B). Although all the presented results were acquired using bovine cyt c,

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qualitatively identical results were also obtained with horse cyt c (not shown).

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The heme moiety elimination/disruption in acyt c and pcyt c was evaluated by EPR spectroscopy at low temperature (Fig. 1C). The peaks of the high spin state of iron can only

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be found for native cyt c in contrast to acyt c and pcyt c, where iron was eliminated. This result is in agreement with the original findings [17]. In addition, the quality of cytochrome c

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samples was confirmed using the liquid chromatography on-line connected with highresolution tandem mass spectrometry technique on the peptide fragment CAQCHTVEK-heme

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(1634.62 Da). This fragment was definitely detected only in native cyt c, but not in the case of

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disrupted versions of the protein in agreement with [27].

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3.2. Electrochemistry and EPR Spectroscopy The above evaluated proteins (in purified monomeric state) were adsorbed for 10 s at the

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surface of the working electrodes, followed by cathodic CPSA and anodic SWV. The ability of cytochromes to form adsorbed layers at mercury and carbon electrodes was previously reported [31, 32]. CPSA was performed at the HMDE, while SWV was done at the PGE. All experiments were carried out using a protein concentration of 1 µM, which is a concentration necessary for full coverage of the electrode surface after 10 s adsorptive accumulation (see Fig. S1 in Supporting Information). The out-of-phase ACV measurements did not show

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT undesirable desorption processes at negatively charged interface in agreement with published results (not shown) [22, 33].

3.2.1. Electrocatalytic and Reduction Processes Cathodic CPS yielded an electrocatalytic signal associated with a catalytic hydrogen evolution

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reaction (CHER) [12, 34, 35]. In this reaction, cyt c acts as a catalyst and produces

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electrocatalytic peak H (around –1.75 V), due to the presence of basic amino acid (aa)

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residues with labile protons, i.e. Lys, Arg and His (Fig. 2A). The results indicate that at slightly acidic and neutral pH, cyt c is an effective catalyst, corresponding well with its basic

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character; pI ~ 10 [36].

Although Cys residues may also participate in CHER [37, 38], the only two Cys

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residues in cyt c are present in the CXXC motif bound to the heme component (Fig. S2 in Supporting Information), and thus do not take part in the CHER. CPS measurement shows an

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absence of electrochemical signal specific to the reduction of Cys residues, i.e. peak S [38], in

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cyt c and pcyt c. However the signal from Cys reduction should be present in acyt c, possessing Cys residues due to the heme elimination [15]. The occurrence of Cys residues in

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acyt c was confirmed using EPR analysis with a Cys-selective spin-probe, 3-maleimidoproxyl (5-MSL) [39]. More specifically, a significantly higher intensity of the spectrum of

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acyt c labeled with 5-MSL (Fig. 3A) indicates that 5-MSL can only bind to free Cys residues in acyt c. Despite the presence of free Cys residues in acyt c, they did not participate in electroreduction processes, which might be explained by localization of the CXXC motif inside a cavity [40, 41] or their general inaccessibility to the electrode surface. The localization of electrochemically active aa residues in cyt c, most likely participating in the CHER, is visualized via surface models in Fig. 3B. In addition to peak H, with cyt c we observed another peak HM at ca. –1.55 V. This peak was, however, not present

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT in acyt c and pcyt c (Fig. 2A, inset). Based on the comparative analysis using hemin (heme analog with a chloride ligand), whose reduction potential was observed at ca. –1.45 V (Fig. 2A, inset), we showed that peak HM is a result of the reduction of the heme component. This was supported by the fact that both heme disrupted analogues did not give peak HM under the same experimental conditions.

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The above described experiments were performed with bare HMDE, which could be

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connected to structural changes of cytochrome c molecules in adsorbed state due to their

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direct contact with Hg-surface. To eliminate or suppress these possible effects, HMDE surface was modified with compact DTT layer according to the previously reported protocol

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[24, 25] prior to cytochrome c adsorption and consequent CPS analyses (Fig. 2B). When comparing cyt c with its structural analogs using electrocatalytic peak H at DTT-modified

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electrode, the disrupted forms (especially acyt c) displayed a slight shift of peak potential to the less negative potentials compared to the cyt c protein. (a) This may be attributed to a

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destabilization of cyt c structure after heme moiety disruption, as indicated by native PAGE

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according to the previously reported protocol (not shown) [26]. The shift of peak H potential to less negative values and increase of height or area of peak H have been observed for other

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structurally destabilized or unfolded proteins [24, 25]. This could be linked to increased accessibility of electrocatalytically active aa residues of unfolded proteins to the electrode

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surface in comparison to native (more compact) protein structures. (b) For exact interpretation of CPS behavior among studied cytochromes, differences in interfacial properties between cyt c and its heme-disrupted analogues have to be taken into account. The differences in interfacial behaviour of cyt c, acyt c and pcyt c were confirmed by phase-sensitive ACV (not shown) and will be, together with other aspects concerning stabilization of the structure of cytochromes in adsorbed state, published elsewhere in more detail.

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ACCEPTED MANUSCRIPT In addition to CPS peak H, peak HM was also observed for cyt c at DTT-modified electrode. Both peak H and peak HM were observed at more negative potentials using DTTmodified electrode in comparison to bare HMDE. CPS response of hemin was not observed for DTT-modified electrode (Fig. 2B, inset), because DTT surface modification prevents lowmolecular compounds to be involved in electrochemical reaction in agreement with literature

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data [24, 25].

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3.2.2. Electrooxidation at Carbon Electrode and Results Applicability

Carbon-based electrodes can be used for monitoring the oxidation of Tyr and Trp within the

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protein structure [13, 42, 43]. As with the cathodic processes described above, these anodic processes primarily involve aa residues localized close to the protein surface and available for

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electron transfer with the electrode surface (Fig. 3B). Cyt c oxidation was manifested by a peak at +0.7 V (Fig. 3C), a result of both Tyr and Trp oxidation merging into this single peak

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(YW peak) at the basal-plane PGE. In addition, cyt c yielded another peak HM’ at +0.95 V

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due to an oxidation of the heme component, as indicated by an absence of this peak in both heme-disrupted analogs. We also confirmed this in the hemin analysis (Fig. 3C). Moreover, a

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similar voltammetric response for cyt c was previously observed with a paraffin-waximpregnated spectroscopic graphite electrode [31].

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Electrocatalytic CPS technique is highly sensitive to protein structural changes, as shown for a diverse spectrum of non-conjugated proteins, e.g. denatured BSA [24, 25]. Here, we report for the first time its application to conjugated protein cyt c and its structural analogs. Recently, CPS was successfully applied for analysis of membrane proteins present in complex lipids or detergents mixtures [44, 45], opening a door for application of the method in studies focusing on cyt c interaction with lipid systems, e.g. negatively-charged cardiolipins [36]. Compared to CPS at HMDE, SWV at carbon electrodes is, with some exceptions [46],

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ACCEPTED MANUSCRIPT rather insensitive for protein structural changes. However, it may serve well for analysis of chemically (e.g. oxidatively) modified proteins and for analysis of protein aging processes [47]. Both techniques, CPS and SWV, could be applied for heme-related analyses, including investigations of protein heme chemical stability or heme interactions with other ligands.

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4. Conclusion

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Hemeproteins and their prosthetic groups are intensively investigated redox-active systems [48]. In this work, we show that their electroanalysis is not limited only to electron transfer

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between the electrode and a central metal atom in the protein’s prosthetic group, but may be

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extended also to study complex redox processes of the heme and for monitoring intrinsic protein electroactivity, both in a single scan. We demonstrated this approach using cyt c and

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its heme-disrupted analogs, acyt c and pcyt c. We believe that the electrochemical and EPR results obtained here may be further applied not only for the study of cyt c, but also other

Acknowledgements

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hemeproteins.

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This work was supported by the Czech Science Foundation (14-08032S), by the MEYS / COST Action EU-ROS, BM1203 (LD14033), by Institutional Funding from Palacky

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University (IGA_LF_2017_011), by the grant MH CZ - DRO (MMCI, 00209805), and by the project MEYS – NPS I – LO1413. We would like to thank Dr. Petra Dvorakova (Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Czech Republic) for mass spectrometry analysis.

Appendix A. Supplementary data Supplementary data to this article can be found online at...

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Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder, Anal. Chim. Acta, 830 (2014) 23-31.

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[34] T. Doneux, V. Ostatna, E. Palecek, On the mechanism of hydrogen evolution catalysis by proteins: A case study with bovine serum albumin, Electrochim. Acta, 56 (2011) 9337-9343. [35] M. Tomschik, L. Havran, M. Fojta, E. Palecek, Constant Current Chronopotentiometric Stripping Analysis of Bioactive Peptides at Mercury and Carbon Electrodes, Electroanalysis, 10 (1998) 403-409. [36] V.E. Kagan, H.A. Bayir, N.A. Belikova, O. Kapralov, Y.Y. Tyurina, V.A. Tyurin, J. Jiang, D.A. Stoyanovsky, P. Wipf, P.M. Kochanek, J.S. Greenberger, B. Pitt, A.A. Shvedova, G. Borisenko, Cytochrome c/cardiolipin relations in mitochondria: a kiss of death, Free Radic. Biol. Med., 46 (2009) 1439-1453.

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ACCEPTED MANUSCRIPT [37] V. Dorcak, V. Ostatna, E. Palecek, Electrochemical reduction and oxidation signals of angiotensin peptides. Role of individual amino acid residues, Electrochem. Commun., 31 (2013) 80-83. [38] V. Dorcak, E. Palecek, Electrochemical Determination of Thioredoxin Redox States, Anal. Chem., 81 (2009) 1543-1548. [39] O.H. Griffith, H.M. McConnell, A nitroxide-maleimide spin label, Proc. Natl. Acad. Sci. USA, 55 (1966) 8-+.

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[40] I. Bertini, J.G. Huber, C. Luchinat, M. Piccioli, Protein Hydration and Location of Water Molecules in Oxidized Horse Heart Cytochrome c by 1H NMR, J. Magn. Reson., 147 (2000)

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1-8.

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[41] C.A. Bortolotti, A. Amadei, M. Aschi, M. Borsari, S. Corni, M. Sola, I. Daidone, The reversible opening of water channels in cytochrome c modulates the heme iron reduction

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potential, J. Am. Chem. Soc., 134 (2012) 13670-13678.

[42] T.A. Enache, A.M. Oliveira-Brett, Peptide methionine sulfoxide reductase A (MsrA): Direct electrochemical oxidation on carbon electrodes, Bioelectrochemistry, 89 (2013) 11-18.

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[43] V. Ostatna, V. Vargova, R. Hrstka, M. Durech, B. Vojtesek, E. Palecek, Effect of His6tagging of anterior gradient 2 protein on its electro-oxidation, Electrochim. Acta, 150 (2014)

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218-222.

[44] J. Vacek, M. Zatloukalova, M. Havlikova, J. Ulrichova, M. Kubala, Changes in the

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intrinsic electrocatalytic nature of Na+/K+ ATPase reflect structural changes on ATP-binding: Electrochemical label-free approach, Electrochem. Commun., 27 (2013) 104-107. [45] M. Zatloukalova, E. Orolinova, M. Kubala, J. Hrbac, J. Vacek, Electrochemical

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Determination of Transmembrane Protein Na +/K +-ATPase and Its Cytoplasmic Loop C45, Electroanalysis, 24 (2012) 1758-1765.

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[46] V. Ostatna, H. Cernocka, K. Kurzatkowska, E. Palecek, Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes, Anal. Chim. Acta, 735 (2012) 31-36.

[47] S. Jaisson, P. Gillery, Evaluation of nonenzymatic posttranslational modification-derived products as biomarkers of molecular aging of proteins, Clin. Chem., 56 (2010) 1401-1412. [48] C.J. Reedy, B.R. Gibney, Heme Protein Assemblies, Chem. Rev., 104 (2004) 617-649.

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ACCEPTED MANUSCRIPT Figure legends:

Fig. 1. (A) UV-Vis absorption spectra of 8 µM bovine cyt c (–––, red line), pcyt c (----, purple line) and acyt c (–––, blue line) in 20 mM Tris-HCl, pH 7.4. Inset: 450-600 nm zoom. (B) SDS-PAGE and Western blot analysis of bovine cytochromes using polyclonal rabbit H-104

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antibody. (C) EPR spectra of 150 µM bovine cytochromes at 19 K.

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Fig. 2. CPS records of 1 µM bovine cyt c (–––, red line), pcyt c (----, purple line), acyt c (–––,

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blue line) and 2.5 µM hemin (–––, pink line) recorded at bare (A) and DTT-modified (B)

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HMDE in 0.1 M Na-phosphate buffer (pH 7); pure supporting electrolyte (–––, black line).

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Fig. 3. (A) EPR spectra of 40 µM bovine cytochromes labeled with 3-maleimido-proxyl (5MSL). (B) Surface models of bovine cyt c (2B4Z) with electroactive aa residues highlighted:

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Tyr (marine blue), Cys (pink), Lys (magenta), His (dark blue), Arg (brown) and Trp (orange).

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(C) SW voltammograms of 1 µM bovine cyt c (–––, red line), pcyt c (----, purple line) and acyt c (–––, blue line) and 1 µM hemin (–––, pink line) recorded in Britton-Robinson buffer

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(pH 6.5) at PGE; pure supporting electrolyte (–––, black line).

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT Figures: Fig. 1.

0.6

0.05 450

500

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Wavelength(nm) λ / nm

0.4

300

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yt c

Pc

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80 0

-80

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Pcyt c

0 -80 1000

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Magnetic field / G

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Ac yt c

Cy tc

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Wavelength(nm) λ / nm

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0 0.10

Cyt c

80

0.15

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EPR signal intensity × 103

Cyt c Pcyt c Acyt c

1.2

A Absorbance

A

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT Fig. 2.

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0.3

500

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750

0.2

0.1

0.0

-1.80

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(dE/dt)

-1

/ s/V

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(dE/dt)-1 / s/V

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250

-1.65

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-1 / s/V (dE/dt)

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Cyt c Acyt c Pcyt c Hemin Blank

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HM 0.10 0.05 0.00 -1.9

-1.8

-1.7

-1.6

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E / V (vs. Ag/AgCl) -1.8

-1.7

-1.6

-1.5

-1.4

E / V (vs. Ag/AgCl)

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT Fig. 3.

A

B

15

5-MSL:

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-15

0

Acyt c

HM’

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-15 5-MSL Cys bound

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YW

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Pcyt c

Free 5-MSL

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0

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EPR signal intensity × 105

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Free 5-MSL

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Cyt c

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3450 3475 3500 3525 3550 3575 0.2

0.4

0.6

0.8

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1.2

E / V (vs. Ag/AgCl)

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Magnetic field / G

5 A

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT

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Graphical abstract

Electroactivity of Cytochrome c and its Structural Analogues

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ACCEPTED MANUSCRIPT Highlights ► The electrochemistry of cytochrome c and its apo and porphyrin analogues is reported. ► Redox behavior of cytochrome c is substantially different to both analogs. ► Specific cathodic/anodic reactions of heme occurred at around –1.55/+0.95 V(vs Ag/AgCl).

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► EPR spin-probe (3-MSL) binding to free Cys residues in apocytochrome c was shown.

Electroactivity of Cytochrome c and its Structural Analogues

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Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes invo...
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