Site-specific cross-linking as a method for studying intramolecular electron transfer.
Site-directed mutagenesis has been used to introduce cysteine residues into yeast cytochrome c peroxidase and yeast cytochrome c for the purpose of forming site-specific cross-linked intermolecular complexes. This enables the formation of well-defined homogeneous covalently linked complexes for the purpose of relating structure to intramolecular electron transfer. Two complexes have been prepared and analyzed. Complex I has an engineered cysteine at position 290 near the C-terminus of the peroxidase linked to the naturally occurring Cys102 near the C-terminus of yeast cytochrome c. This complex exhibits undetectable rates of intramolecular electron transfer. Complex II has Cys290 of the peroxidase linked to the engineered Cys73 of cyt c. This complex was designed to mimic the crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. Stopped-flow studies show that complex II carries out intramolecular electron transfer from ferrocytochrome c to peroxidase compound I at a rate of approximately 500-800 s-1. This indicates that the binding orientation observed in the crystal structure is competent in rapid intramolecular electron transfer.
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Department of Molecular Biology, University of California, Irvine 92717, USA.
Poulos
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D001483:Base Sequence; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D003578:Cytochrome-c Peroxidase; D003545:Cysteine