The goal of the proposed research is to elucidate structural determinants important for efficient electron transfers between the FMN- and heme- containing domains of flavocytochrome b2, and between the heme-domain and cytochrome c. This understanding will aid future attempts to engineer redox proteins containing novel combinations of redox centers. Flavocytochrome b2 is an ideal model for investigation of inter-domain electron transfers by site-directed mutagenesis as the x-ray structure is known to 2.4 A resolution and fully-active plasmid-encoded enzyme can be expressed in high yield E. coli. The FMN- and heme-centers exhibit common structural and mechanistic features with a large number of related metalloproteins. The specific objectives are to introduce selected mutations at sites expected to modify (i) specific interactions between the FMN domain and heme, (ii) the heme redox potential , and (iii) interaction between flavocytochrome b2 and cytochrome c. The resulting perturbations of FMN and heme redox properties will be determined by CD, visible and EPR potentiometry. Rates of intramolecular electron transfer, associated enthalpies and cooperative redox interactions between FMN and heme will be determined by redox-controlled T-jump relaxation and laser-flash photolysis. Intermolecular electron transfer to cytochrome c will be studied using stopped-flow spectrophotometry. Steady-state kinetic analysis will identify changes in substrate and produce binding and in catalytic efficiency. Controls for gross structural perturbation due to mutations will include CD spectroscopy and urea denaturation studies. Crystallization and x-ray analysis of mutants of particular interest will be attempted to obtain their structures from difference Fourier maps. Results will be interpreted in the light of the structures of flavocytochrome b2 and cytochrome c, current theories of biological electron transfers and models of electrostatic interactions within proteins.
| Kay, C J; Lippay, E W (1992) Mutation of the heme-binding crevice of flavocytochrome b2 from Saccharomyces cerevisiae: altered heme potential and absence of redox cooperativity between heme and FMN centers. Biochemistry 31:11376-82 |
