EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Summary

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

Abstract

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.

The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.

A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

Introduction

It is striking that the most fundamental chemical processes sustaining life on this planet, photosynthesis, nitrogen fixation and respiration, are catalyzed by large protein complexes containing a wide range of organic and inorganic redox cofactors. It has been estimated that approximately 30% of all proteins contain one or more metal cofactors.^1,2 Identifying and characterizing the redox cofactors can be established using direct electrochemistry (e.g., protein film voltammetry) or redox titrations. The two techniques are complementary in their nature and applicability. Voltammetry offers fast determination of midpoint potentials and electron trans....

Protocol

1. Preparation of Setup and Solutions

1. Prepare buffer solution containing 100 mM Tris, 250 mM NaCl and 10% glycerol at pH 8.5. Deaerate the buffer by flushing with argon. Introduce the buffer into an anaerobic chamber.
2. Divide the buffer in two portions. To one portion (buffer A) add 1 mM DL-dithiothreitol (DTT, M_r 154.25 g/mol) and 1 mM L-cysteine. The other portion (buffer B) does not contain reducing agents.
3. Prepare oxidant and reductant solutions. Make 1 ml solutions of 2, 20, and 200 mM potassium ferricyanide (FIC, M_r 329.26 g/mol) and of sodium dithionite (SD, M_r 174.11 g/mol) in anaerobic buffer B.<....

Results

The redox titration of the iron-sulfur cluster containing protein Nar1 from Saccharomyces cerevisiae resulted in the identification of three different cofactors: Nar1: a [3Fe-4S] cluster, a [4Fe-4S] cluster and a mononuclear Fe center (Figure 1). The EPR signals are characterized by their g-values: for the [3Fe-4S]⁺ g_z 2.01, g_crossover 2.00 (Figure 1B); for the [4Fe-4S]⁺, g_z 2.02 g_y 1.93, g_x 1.82 (

Discussion

The series of 13 redox mediators allows a redox equilibrium in the solution in the range from -0.45 to +0.3 V versus SHE (Table 1 and Figure 3). Well above and below these potentials all mediators are either completely reduced or completely oxidized and hence no further equilibration with the redox active centers of the protein can occur. This is an important notion, as in literature sometimes redox titrations are performed with only two or three mediators, allowing only a narrow range t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reference electrode (Ag/AgCl)	Radiometer
Chemicals	Sigma-Aldrich
EPR spectrometer	Bruker
N,N,N’,N’-tetramethyl-p-phenylendediamine (TMPD) · (HCl)2			637-01-4
2,6-dichlorophenol indophenol (DCIP), sodium salt			620-45-1
Phenazine ethosulfate (PES)			10510-77-7
Methylene blue			122965-43-9
Resorufin, sodium salt			34994-50-8
Indigodisulfonate (indigo carmine)			860-22-0
2-hydroxy-1,4-naphtaquinone			83-72-7
Anthraquinone-2-sulfonate Na+ H2O			153277-35-1
Phenosafranin			81-93-6
Safranin O			477-73-6
Neutral red			553-24-2
Benzyl viologen			1102-19-8
Methyl viologen			1910-42-5