Investigations of Yeast L-Lactate Dehydrogenase (Cytochrome b2)

The optical rotatory dispersion of crystalline cytochrome b2, the l(+)-lactate dehydrogenase (EC 1.1.2.3) of bakers' yeast, is observed in the wave length range, 600 to 195 mµ. The dispersion at short wave lengths is similar in character to that observed with α-helical polypeptides, including a shoulder at about 210 mµ, except that the minimum occurs at 230 mµ instead of 233 mµ. The depth of this minimum and the height of the maximum at 199 mµ agree in suggesting that the content of α helix ranges from 15% to 40%, the higher values being observed with more active samples of enzyme. The optical rotatory dispersion in this region is little if at all affected by changes in the oxidation states of the heme and flavin mononucleotide prosthetic groups or by removal of the latter group. Anomalies in the dispersion are observed in the region of the α, β, and γ absorption bands of the heme group. The oxidized enzyme shows a small, negative Cotton effect centered near the γ, or Soret, band, and some small irregularities in the 500 to 600 mµ region. In contrast, the reduced enzyme shows a much larger, negative Cotton effect at the Soret band and a smaller, unsymmetrical one at the α-band (550 mµ). The amplitude of the Soret Cotton effect is inversely dependent on the concentration of l-lactate when this is used as reducing agent. The amplitude of this anomaly is somewhat larger when the enzyme is reduced by mercaptoethanol than when it is reduced by l-lactate or by hydrogen in the presence of a platinum catalyst. The amplitude of the anomaly in the dispersion of the enzyme reduced by l-lactate near the α absorption band is found to be dependent on the concentration of the enzyme in a manner consistent with the view that it results from aggregation. On the other hand, this anomaly is not observed when hydrogen plus catalyst or mercaptoethanol is used as reducing agent. Large changes in the heme Cotton effects result from removal of the FMN prosthetic group. The oxidized apoenzyme has a small, positive effect in the Soret region, and the reduced apoenzyme has a large, positive effect. These changes are nearly completely reversed by addition of FMN to the apoenzyme; with apoenzyme which was 50% reactivated by addition of FMN, the regenerated Soret Cotton effect had an amplitude 70% of that of the original holoenzyme. The addition of excess p-chloromercuribenzenesulfonate to the reduced enzyme gives rise to a Cotton effect similar to that of the reduced apoenzyme. If the system is kept strictly anaerobic, addition of mercaptoethanol completely reverses this effect. The observations reported here show that the FMN is located close enough to the heme to affect the symmetry of the absorption bands of the latter. The fact that the action of p-chloromercuribenzenesulfonate on the enzyme produces optical rotatory dispersion changes very similar to those resulting from complete removal of FMN suggests that the asymmetry produced by FMN is the result of a significant coupling between the two groups which is weakened by p-chloromercuribenzenesulfonate to the point that it completely loses its effect on the heme absorption bands.