An electrochemical study of the factors responsible for modulating the reduction potential of putidaredoxin.
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 x 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 degrees ', 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 degrees ' of cytochrome P450(cam)-bound putidaredoxin (+44 mV) with respect to the E degrees ' measured for free putidaredoxin.
Avila
L
L
Department of Chemistry, Oklahoma State University Stillwater, OK 74078-3071, USA.
Wirtz
M
M
Bunce
R A
RA
Rivera
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004563:Electrochemistry; D004579:Electron Transport; D004926:Escherichia coli; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011107:Polylysine / Q000737:chemistry
D005288:Ferredoxins; D011107:Polylysine; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System