Investigations of yeast L-lactate dehydrogenase (cytochrome b2). VI. Circular dichroism of the holoenzyme.

Abstract The circular dichroism spectra of oxidized and reduced cytochrome b2, the l(+)-lactate dehydrogenase (EC 1.1.2.3) of bakers' yeast, are observed in the wave length range 188 to 600 mµ. The spectrum at short wave lengths, which is unaffected by change in the oxidation state of the enzyme, is similar in character to that observed with α-helical polypeptides, the amplitudes of the extrema indicating an apparent helical content of roughly 25%. Between 250 and 600 mµ numerous dichroic bands are observed. Type I (DNA-containing) enzyme has a large positive band at 282 mµ because of the DNA component, and Type II (DNA-free) enzyme has a prominent negative band at 270 mµ which appears to result from an interaction of the flavin prosthetic group with 1 or more aromatic residues since it is entirely missing from FMN-free apoenzyme. Neither of these bands is much affected by the oxidation state of the enzyme. All the bands above 300 mµ are strongly dependent on the oxidation state of the enzyme, but are affected very little by removal of the DNA. These bands can be ascribed to transitions in the flavin and heme prosthetic groups, and many of them appear to be strongly affected by the flavin-heme interaction which also appears to be essential for enzymic activity. Numerical integration over the circular dichroism spectrum according to the Kronig-Kramers equation gives a calculated optical rotatory dispersion spectrum which reproduces all the features of the observed spectrum but falls below it in amplitude. A Lowry plot of the difference between the observed and calculated spectra suggests that a dichroic band or bands centered at 150 mµ, the result of peptide units in α-helical conformation, contributes significantly to the dispersion over the entire wave length range studied. The effects of urea and changes of pH on the circular dichroism and activity of the enzyme have been studied. A remarkable parallelism between the amplitude of the dichroic band in the region of the heme Soret band and the activity of the enzyme is observed. At a urea concentration of approximately 3 m (neutral pH) and a pH of 9.5 (no urea present), the dichroic spectrum of the enzyme is similar to that observed for the FMN-free apoenzyme and the enzyme is inactive. These observations give further support to the view that interaction of some sort between the FMN and heme groups is responsible for both the character of the circular dichroism spectrum and the catalytic activity. The circular dichroism of a heme peptide, with a molecular weight of about 12,000, obtained by tryptic digestion of cytochrome b2, is found to be similar to that observed for the FMN-free apoenzyme in the wave length region below 250 mµ and in the region of the Soret band. It is interesting that the apparent α-helical content is roughly the same as for the native enzyme. The cytochrome b2 obtained from the yeast Hansenula anomala, which has an activity approximately 6 times that of the enzyme from Saccharomyces cerevisiae, is found to have circular dichroism spectra similar to those observed with the Type II enzyme below 300 mµ, but differing markedly at higher wave lengths, especially in the Soret region. The difference dichroic spectra of Hansenula enzyme minus Saccharomyces enzyme consist largely of a symmetrical positive band at 423 mµ for the reduced enzymes and one at 413 mµ for the oxidized enzymes. It appears that these bands do not involve the FMN-heme interaction responsible for enzyme activity.