Characterization of redox proteins using electrochemical methods

M. Verhagen

Research output: Thesisinternal PhD, WU


<p>The use of electrochemical techniques in combination with proteins started approximately a decade ago and has since then developed into a powerfull technique for the study of small redox proteins. In addition to the determination of redox potentials, electrochemistry can be used to obtain information about the kinetics of electron transfer between proteins and about the dynamic behaviour of redox cofactors in proteins. This thesis describes the results of a study, initiated to get a better insight in the conditions necessary to obtain electron transfer between solid state electrodes and proteins.<p>Flavin Adenine Dinucleotide (FAD) is the subject of chapter 2. The electrochemical behavior of this cofactor, which is present in some flavoproteins, appeared to be dependent on its solution concentration. At concentrations of 1 μM the voltammetry showed all the characteristics of a species adsorbed to the surface. At a thousandfold higher concentration the voltammetry became completely diffusion controlled. From experiments at intermediate concentrations it was concluded that part of the FAD molecules adsorb to the electrode. Furthermore, it was shown that electron transfer between the molecules in solution and the electrode can only take place through the adsorbed molecules, which act as mediators. A comparison with results obtained with a 2 [4Fe-4S] ferredoxin from <em>Megasphaera elsdenii</em> suggested that, under certain conditions, a similar mechanism of selfmediation can be valid for proteins.<p>The results of a study of cytochrome <em>c</em> 553 from <em>D. vulgaris</em> (H) <em></em> are presented in chapter 3. The cytochrome was characterized by cyclic voltarnmetry and the same technique was used to determine the rate of electron transfer between the cytochrome and the Fe-hydrogenase from the same organism. The results indicated that the cytochrome was a physiologically competent redox partner dependent on the in vivo function of the hydrogenase. Since the function of the hydrogenase is still an issue of debate it is not known whether this new electron transfer pathway has physiological relevance.<p>The reinvestigation of the protein desulfoferrodoxin from <em>D. vulgaris</em> (H) is described in chapter 4. This protein was reported to contain two iron atoms one of which was coordinated by four cysteine residues in a distorted tetrahedron. By comparison with model compounds using EPR spectroscopy and by using cyclic voltarnmetry at different pH values it was shown that this is very unlikely. Instead it is proposed that the iron atom is coordinated in a pentagonal bipyramid (surrounded by 5 ligands in a plane and 2 ligands perpendicular to and on both sides of this plane). Furthermore the controversy about the protein having a mixed N-terminus was elucidated and it was established that the protein was a homodimer instead of the reported monomer.<p>The conditions necessary for the use of direct electrochemistry to study small redox proteins become more and more established. The application of this technique to enzymes is, however, not straightforward, The reason for this is not clear, but one possibility is that a large enzyme adsorbs more easily to the electrode than a small protein. Another possible explanation is that the redox cofactors in enzymes are shielded more by the protein matrix. In order to circumvent this latter problem we tried to establish conditions for the electron transfer between cytochrome P- 450 from <em>P. putida</em> and glassy carbon electrodes. This bacterial cytochrome P-450 has a ferredoxin as a physiological electron donor and has therefore a docking place where the electrons can enter the enzyme. When using the right conditions it should be possible to let the electrode be the "substrate" for the enzyme. Unfortunately the enzyme adsorbed to the electrode and the obtained value for the potential was much more positive than the literature value. An EPR redox titration of the cytochrome P-450 indicates that the literature value needs a correction. However, there still remains a difference between the value obtained from the titration in homogeneous solution and the value determined electrochemically.<p>Recent reports about electrochemical characterization of superoxide dismutase from bovine erythrocytes incited the study described in chapter 6. The conditions used in the reported electrochemical experiments were rather extreme <u>i.e.</u> low pH. EPR monitored redox titrations of the enzyme at different pH values indicated that the oxidation and reduction at low pH values is not reversible. Furthermore, it was found that the reported potentials at pH 7.0 needed to be corrected. A redox titration was also performed with the iron enzyme from <em>E. coli</em> as a comparison with the copper zinc containing enzyme. After reduction, however, it was not possible to reoxidize the enzyme again indicating that the redox reaction is not reversible. This can explain the huge differences in potentials reported so far in the literature.<p>The use of a redox active promotor can give some insight in its mechanism of action. The lanthanide europium proved to be a potent promotor of rubredoxin. The latter is a small purple redox protein containg a single iron coordinated to 4 cysteine residues. At high pH values the reduction and oxidation of rubredoxin is readily obtained despite the fact that the europium ion does not show any reduction or oxidation anymore. This is not consistent with the models used to explain the promotor function of cations. These models all assume that the cation provides charge compensation and sandwiches both between the protein and the electrode as well as between different protein molecules. The results from this study are presented in chapter 7.<p>A great advantage of electrochemistry using glassy carbon electrodes is that it is possible to vary the potential between approximately -1 V and +0.8 V. This makes it possible to study redox couples with potentials more negative than the commonly used chemical reductants like dithionite or titanium citrate. This led to the discovery of the superreduction of the Rieske cluster in the soluble fragment of the <em>bc1</em> complex of beef heart as described in chapter 8. This protein contains an [2Fe- 2S] cluster with a redox potential of + 312 mV versus SHE. At low potential (-840 mV versus SHE) it is possible to reduce this cluster with a second electron. The physiological relevance of this superreduced state is not clear but its characterization can give insight in the mechanism of multiple electron transfer by iron sulfur clusters.<p>The final two chapters are used to describe the biochemical and spectroscopic characterization of two proteins from <em>D. vulgaris</em> . Adenylyl sulphate reductase <em>(AdoPSO <sub>4</sub></em> reductase) is an enzyme which is involved in the sulfate respiration of the bacterium. It reduces the activated sulfate <em>(AdoPSO <sub>4</sub></em> ) to AMP and sulfite. Literature reports indicated that the protein contained one FAD and two [4Fe-4S] clusters. The presence of two clusters was based on the observation of a complicated EPR spectrum which indicates interaction between two paramagnetic centers. In our studies however this "interaction" spectrum only appeared when the enzyme solution was at low ionic strength. Upon raising the ionic strength with an inert salt like NaCl the complicated EPR spectrum changed into a spectrum of a single S-1/2 species. This indicated that the interaction between the paramagnetic centers was intermolecular rather than intramolecular. This observation led us to propose that <em>AdoPSO <sub>4</sub></em> reductase contains one FAD and one Fe-S cluster. Since the average metal analysis showed the presence of 6 iron atoms and 5 acid labile sulfur atoms it was proposed that the Fe-S cluster may have an iron content greater than 4.<p>Chapter 10 describes the results of a study of high molecular weight cytochrome <em>c</em> . This protein resides in the periplasmic space of <em>D. vulgaris</em> and contains sixteen hemes. Its function is up till now unknown. In previous reports midpoint potentials were reported for the different hemes based on single scan differential pulse voltammetry. These values might be erroneous due to the absence of reversibility. Indeed an equilibrium redox titration monitored by EPR indicated that the reported values were incorrect. Furthermore, it was not possible to reproduce the reported voltammograms. This confirmed our observation that the electrochemistry of large proteins or enzymes is often difficult to interpret and difficult to reproduce. It is also a good example of how important it is to check whether or not reversibility applies during electrochemical experiments.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Veeger, C., Promotor
  • Hagen, W.R., Promotor, External person
Award date1 May 1995
Place of PublicationS.l.
Print ISBNs9789054853800
Publication statusPublished - 1995


  • oxidoreductases
  • cytochromes
  • electrochemistry

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