Sulfite binding to a flavodehydrogenase, cytochrome b2 from baker's yeast

Area of Science:

• Biochemistry
• Enzymology
• Protein Structure and Function

Background:

• Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a flavodehydrogenase with a monoelectronic acceptor.
• It exhibits a red semiquinone form and interacts with various substrates and inhibitors.

Purpose of the Study:

• To investigate the interaction of flavocytochrome b2 with sulfite.
• To characterize the formation and properties of a flavocytochrome b2-sulfite complex.
• To elucidate the role of active site charges in enzyme function and substrate binding.

Main Methods:

• Difference spectroscopy was employed to observe the flavin chromophore.
• Enzymatic assays were conducted to study inhibition kinetics.
• Binding studies were performed to determine the dissociation constant (Kd) for the sulfite complex.

Main Results:

• A reversible covalent complex between flavocytochrome b2 and sulfite was identified with a dissociation constant (Kd) of 1.4 muM.
• Difference spectroscopy enabled visualization of the flavin chromophore, overcoming interference from heme absorbance.
• Anions like D-lactate, oxalate, and pyruvate were found to be inhibitors, inducing spectral changes in the flavin.

Conclusions:

• The active site likely possesses two positive charges: one stabilizing the red semiquinone and another binding organic anions and sulfite.
• The formation of a covalent sulfite complex provides a tool for studying the flavin environment.
• The correlation between sulfite and oxygen reactivity in flavoproteins may be less general than previously thought.