Site-directed mutagenesis of rubredoxin reveals the molecular basis of its electron transfer properties.
Rubredoxins contain a single non-heme iron atom coordinated by four cysteines. This iron is redox active and confers a role to these proteins in electron transfer chains. The structural features responsible for setting the values of the reduction potential and of the electron self-exchange rate constant have been probed by site-directed mutagenesis. Replacements of the highly conserved residues in positions 8, 10, and 11 (valine, glycine, and tyrosine, respectively) all lead to shifts of the reduction potential, up to 75 mV. These cannot be explained by simple considerations about the physicochemical properties of the substituting side chains but rather indicate that the value of the reduction potential is finely tuned by a variety of interactions. In contrast, the electron self exchange rate constant measured by nuclear magnetic resonance does not vary much, except when a charged residue is included in position 8 or 10, at the surface of the protein closest to the iron atom. Analysis of the data with a model for electrostatic interactions, including both monopolar and dipolar terms, indicates that the presence of a charge in this region not only increases the repulsion between molecules but also affects the electron transfer efficiency of the bimolecular complexes formed. The studies presented constitute a first step toward probing the structural elements modulating the reactivity of the FeS4 unit in a protein and defining the electron transfer active site(s) of rubredoxin.
Kümmerle
R
R
CEA, Département de Recherche Fondamentale sur la Matière Condens-ee, SCIB/SCPM, 38054 Grenoble Cedex 9, France.
Zhuang-Jackson
H
H
Gaillard
J
J
Moulis
J M
JM
D001665:Binding Sites; D003013:Clostridium / Q000737:chemistry*; D003198:Computer Simulation; D004563:Electrochemistry; D004579:Electron Transport; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011199:Potentiometry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012416:Rubredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013696:Temperature; D014867:Water / Q000378:metabolism
D011994:Recombinant Proteins; D012416:Rubredoxins; D014867:Water