Determination of cytochrome b5 association reactions. Characterization of metmyoglobin and cytochrome P-450(cam) binding to genetically engineered cytochrome

P. S. Stayton, M. T. Fisher, S. G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

Genetically engineered cytochrome b5 has been used to quantitate binding interactions of this protein with cytochrome P-450(cam) 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 Å, 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 metmyglobin, cytochrome c, and cytochrome P-450(cam), whereas the acrylodan-labeled T8C mutant fluorescence emission remained unchanged during all titrations. Dissociation constants of 1.3, 0.6, and 0.5 μM, pH 7.15, were measured for metmyglobin, cytochrome P-450(cam), and cytochrome c, respectively. A similar averaged binding surface for cytochrome P-450(cam) 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.

Original languageEnglish (US)
Pages (from-to)13544-13548
Number of pages5
JournalJournal of Biological Chemistry
Volume263
Issue number27
StatePublished - 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Determination of cytochrome b<sub>5</sub> association reactions. Characterization of metmyoglobin and cytochrome P-450(cam) binding to genetically engineered cytochrome'. Together they form a unique fingerprint.

Cite this