Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding.
Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Walker
M C
MC
Department of Biochemistry, University of Arizona, Tucson 85721.
Tollin
G
G
D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007834:Lasers; D008024:Ligands; D010084:Oxidation-Reduction; D010782:Photolysis; D010843:Pichia / Q000201:enzymology*; D011773:Pyruvates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D008024:Ligands; D011773:Pyruvates; D006418:Heme; D005486:Flavin Mononucleotide; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)