Pyridine‐Nucleotide Transhydrogenase

Abstract

From spectral titrations of oxidised transhydrogenase from Azotobacter vinelandii with NADP + the existence of at least one NADP + complex is clearly demonstrated. Complex formation is responsible for the changes in the visible region of the absorption spectrum, the quenching of the flavin fluorescence and the shift of the fluorescence emission maximum. Moreover, the complex with the highest affinity is responsible for the changes in the optical‐rotatory‐dispersion and circulardichroism characteristics. The dissociation constant ( K d ) of this NADP + complex is 3–4 μM. Changes in the 300–400 nm region of the absorption spectrum at higher NADP + concentrations point to the existence of a second binding site for NADP + with a much lower affinity. Several proposals to explain shifts in flavin absorption like changes in polarity around the flavin, or interaction between NADP + and the isoalloxazine ring of the flavin, or weakening of ribityl side chain interactions, or the possible involvement of protein conformational changes are discussed.