CHANGES IN SECONDARY STRUCTURE AND FLAVIN MICROENVIRONMENT BETWEEN AZOTOBACTER-VINELANDII LIPOAMIDE DEHYDROGENASE AND SEVERAL DELETION MUTANTS FROM CIRCULAR-DICHROISM

Circular 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 Delta 9 and Delta 14). From analysis of CD-spectra in the 190-240 nm region it was found that the alpha-helix content did not change (32%) among the three proteins, but the beta-sheet structure is distinctly less in case of the deletion mutants as compared to the wild-type enzyme. Upon dilution of the 414 mutant from 30 mu M to 2 mu M the CD spectrum showed a drastic reduction in alpha-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 Delta 9 mutant) and the Delta 14 mutant. While wild-type and Delta 9 mutant proteins have virtually no optical activity within the lowest energy absorption band, the Delta 14 mutant gives a negative Cotton effect in this region. The second absorption band at higher energy shows strong positive optical activity in case of Delta 9 mutant and wild-type enzyme and a smaller effect in case of the Delta 14 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 Delta 14 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).