Changes in secondary structure and flavin microenvironment between Azotobacter vinelandii lipoamide dehydrogenase and several deletion mutants from circular dichroism

AbstractCircular dichroism (CD) has been used to investigate the secondary structure of wild-type lipoamide dehydrogenase from Azotobacter vinelandii and two deletion mutants lacking 9 and 14 C-terminal amino acids, respectively (these mutants are referred to as Δ9 and Δ14). From analysis of CD-spectra in the 190–240 nm region it was found that the α-helix content did not change (32%) among the three proteins, but the β-sheet structure is distinctly less in case of the deletion mutants as compared to the wild-type enzyme. Upon dilution of the Δ14 mutant from 30 μM to 2 μM the CD spectrum showed a drastic reduction in α-helix content (from 32% to 17%) which is ascribed to a weakening of the subunit-subunit interaction. The region where the flavin prosthetic group absorbs light (300–500 nm) exhibits characteristic changes between wild-type protein (or Δ9 mutant) and the Δ14 mutant. While wild-type and Δ9 mutant proteins have virtually no optical activity within the lowest energy absorption band, the Δ14 mutant gives a negative Cotton effect in this region. The second absorption band at higher energy shows strong positive optical activity in case of Δ9 mutant and wild-type enzyme and a smaller effect in case of the Δ14 mutant. Since the optical activity of the flavin chromophore in flavoproteins is very sensitive to its microenvironment, the experimental results are indicative of a changed surrounding of the flavin, which is in full agreement with the partial exposure of the flavin to the solvent in the Δ14 mutant (in contrast to the shielded flavin in wild-type enzyme), as found previously with fluorescence relaxation spectroscopy (Bastiaens, P.I.H., Van Hoek, A., Van Berkel, W.J.H., De Kok, A. and Visser, A.J.W.G. (1992) Biochemistry 31, 7061–7068)