Determination of cytochrome b5 association reactions. Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochromeb5.
Genetically engineered cytochrome b5 has been used to quantitative binding interactions of this protein with cytochrome P-450cam and sperm whale metmyoglobin by static fluorescence titration. Two cytochrome b5 mutants were constructed by cassette mutagenesis to replace a surface threonine residue with cysteine at two crystallographically defined positions, 65 and 8, located 11 and 21 A, respectively, from the nearest heme edge. The T65C and T8C mutant proteins were labeled with the sulfhydryl selective fluorescent reagent, acrylodan, which provided a spectral probe for monitoring protein-protein association. The fluorescence emission spectra of the acrylodan-labeled T65C mutant exhibited an ionic strength-dependent, blue-shifted fluorescence enhancement upon binding met-myoglobin, cytochrome c, and cytochrome P-450cam, whereas the acrylodan-labeled T8C mutant fluorescence emission remained unchanged during all titrations. Dissociation constants of 1.3, 0.6, and 0.5 microM, pH 7.15, were measured for metmyoglobin, cytochrome P-450cam, and cytochrome c, respectively. A similar averaged binding surface for cytochrome P-450cam and cytochrome c is suggested by their closely related degree of fluorescence enhancement, degree of emission blue shift, and binding free energies. Myoglobin binds less tightly, enhances fluorescence to a greater extent, and exhibits a larger blue shift in acrylodan emission spectra suggesting a different averaged binding orientation relative to the acrylodan probe.
Department of Biochemistry, University of Illinois, Urbana 61801.
P S
PS
Stayton
M T
MT
Fisher
S G
SG
Sligar
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5; D004579:Electron Transport; D005456:Fluorescent Dyes; D006420:Hemeproteins / Q000378:metabolism*; D008786:Metmyoglobin / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009154:Mutation; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D014907:Whales
D003573:Cytochrome b Group; D005456:Fluorescent Dyes; D006420:Hemeproteins; D011994:Recombinant Proteins; D008786:Metmyoglobin; C038518:acrylodan; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D015081:2-Naphthylamine; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase