An Electrochemical Study of the Factors Responsible for Modulating the Reduction Potential of Putidaredoxin |
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Authors: | Ludivina Avila Marc Wirtz Richard A Bunce Mario Rivera |
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Institution: | (1) Department of Chemistry, Oklahoma State University Stillwater, OK 74078–3071, |
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Abstract: | The gene coding for putidaredoxin has been synthesized using a combination of chemical and enzymatic methods and subsequently
expressed in Escherichia coli. The recombinant protein characterized by electronic spectroscopy, mass spectrometry, and electrochemistry was found to be
identical to putidaredoxin obtained from Pseudomonas putida. Polylysine was found to promote the fast and reversible electrochemistry of putidaredoxin at negatively charged electrodes
such as indium-doped tin oxide or gold surfaces modified with mercaptoalkanoate groups. The value of the heterogeneous electron
transfer rate constant obtained from solutions containing a mixture of putidaredoxin and polylysine (k
s
=1.3×10–3 cm/s) is one order of magnitude larger than the values reported previously at gold electrodes modified with mercaptoethylamine
or at antimony-doped tin oxide semiconductor electrodes. It was observed that when the reduction potential of putidaredoxin
is measured by cyclic voltammetry, the resultant value is consistently more positive (64 mV) than the reduction potential
measured with potentiometric titrations. A comparison between the electrochemical responses of putidaredoxin and spinach ferredoxin,
combined with the examination of their corresponding three-dimensional structures, indicates that the positive shift in the
reduction potential of putidaredoxin originates from the formation of a transient complex between putidaredoxin and polylysine
at the electrode surface. The formation of this transient complex modulates the reduction potential of putidaredoxin by lowering
the value of the dielectric constant around its iron-sulfur cluster microenvironment, specifically by neutralizing negative
charges surrounding the active site and by excluding water from the solvent exposed iron sulfur cluster. The observed positive
shift in E°′, which is induced by complexation with polylysine at the electrode-surface, suggests that similar factors are
likely to contribute to the anodic shift in the E°′ of cytochrome P450cam-bound putidaredoxin (+44 mV) with respect to the E°′ measured for free putidaredoxin.
Received: 14 June 1999 / Accepted: 6 August 1999 |
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Keywords: | Putidaredoxin Electrochemistry Cyclic voltammetry Direct electrochemistry Cytochrome P450 Reduction potential |
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