Studies on Lipoamide Dehydrogenase of Bakers' Yeast:V. The Effects of Metal Ions and Sulfhydryl Reagents on the Absorption Spectrum of the Enzyme

The changes in the absorption spectrum of lipoamide dehydrogenase from Saccharomyces oviformis were investigated on addition of excess NADH₂ in the presence of various metal ions, arsenite or PCMB under anaerobic conditions.<br> With group I metal ions (Fe⁺⁺ and Co⁺⁺), the flavin moiety of the enzyme is reduced by NADH₂ to a red-intermediate and is oxidized in air to a yellow form. This suggests that the metal combines weakly with the dithiol group of the enzyme and is split from the enzyme protein by reoxidation in air. With group II metal ions (Zn⁺⁺, Cd⁺⁺ and Cu⁺⁺), the enzyme is reduced by NADH₂ to a green form via a red-intermediate and shows a new absorption band at a longer wavelength. On reoxidation in air, the green form changes to a yellow form, the spectrum (band around 450 mμ) of which is shifted to a shorter wavelength. After BAT.L treatment, the spectrum returns to that of native enzyme. Arsenite also combines with the dithiol group of the enzyme which has been fully reduced by NADH₂ via a red-intermediate. The green form shows a new absorption band at a longer wavelength and is reoxidized in air to the yellow oxidized enzyme, suggesting the dissociation of arsenite from the enzyme protein. Like native enzyme, PCMB-treated enzyme in which free -SH groups are blocked, is reduced by NADH₂ to a red-intermediated. This PCMB-enzyme is reduced by NADH₂ in the presence of PCMB to a colorless form via a red-intermediate. It has no absorption band at a longer wavelength. It changes to a yellow oxidized form in air, with a spectrum shifted to shorter wavelengths. This reoxi-dized enzyme is directly reduced by NADH₂ to the colorless form. In the presence of Cd⁺⁺, the PCMB -enzyme is reduced by NADH₂ to a green form via a red-inter-mediate. The spectrum of the green form shows no absorption band at a longer wave-length. On reoxidation in air, the green color changes to yellow and the spectrum shifts to a shorter wavelength. The reoxidized enzyme can be directly reduced by NADH₂ to the green form without passing through a red-intermediate.<br> The authors wish to thank Dr. G. Sunagawa, the Director of this Laboratory, for his encouragement during the investigation.