Cytochrome b562 variants: a library for examining redox potential evolution.
A general understanding of how cytochromes evolve within a fixed structure to optimize redox potential for specific bioenergetic processes does not exist. Toward this end, a library approach is used to investigate the range and distribution of redox potential which occurs when all sequence space available through mutation at two positions is examined within a fixed structural motif. Random mutation of Phe61 and Phe65 of cytochrome b562 (E. coli), and subsequent examination of a statistically significant sampling of this library, demonstrates that the redox potential can vary over 100 mV (>25% of the known accessible potential in native proteins with axial His-Met ligation) through mutation at these two positions. The redox potential of the wild-type protein occurs at an extremum of the distribution observed, indicating that Phe61 and Phe65 were most likely naturally selected to differentially stabilize the reduced state of the protein. At the other extremum, a compositionally conservative set of mutations (F61I, F65Y) leads to a 100 mV shift in the redox equilibrium toward the oxidized state. NMR analyses indicate that a charge-dipole interaction which results from mutation of phenylalanine to tyrosine at position 65 may be responsible.
Springs
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SL
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Bass
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SE
McLendon
G L
GL
D019943:Amino Acid Substitution / Q000235:genetics; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D019143:Evolution, Molecular; D014644:Genetic Variation; D016254:Mutagenesis, Insertional; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D019151:Peptide Library; C017850:cytochrome b562, E coli