Étude par dichroïsme circulaire des interactions hème‐flavine‐protéine dans le cytochrome b2 ( l ‐lactate oxydoréductase) de la levure

The circular dichroism spectra of bakers' yeast l‐lactate dehydrogenase and some of its derivatives (without flavin and/or heme) have been investigated in order to obtain some information about the interactions between the prosthetic chromophores or between these chromophores and the protein moiety. The following derivatives were studied: (a) the cytochrome b2 (flavohemoprotein), (b) the apocytochrome b2 (following FMN removal), (c) the noyau cytochromique b2 (low molecular weight core isolated following proteolytic degradation), (d) the apo‐noyau cytochromique b2 (obtained by removal of the heme from the latter). It is shown that the circular dichroism spectra of the protein moieties in the ultraviolet region (200–250 nm) are not affected, either by the removal of the flavin from the flavocytochrome b2, or by the removal of the heme from the noyau cytochromique b2. The significance of the values calculated for the α‐helical content is discussed. The oxido‐reduction state of the ferric ion does not affect the circular dichroism spectra in this region. The circular dichroism bands associated with the heme chromophore are essentially the same in the apocytochrome b2 and in the noyau cytochromique b2, i. e. both without the flavin moiety, but are markedly changed in the flavocytochrome b2, in particular with an inversion of the strong γ band. Noticeable differences are observed with a homologous enzyme extracted from the yeast Hansenula anomala, especially in the γ band region. In conclusion there appears to be no prosthetic group‐protein interaction detectable by circular dichroism measurements, i. e. no important modification of the peptide bond geometry occurs when the flavin is removed from the enzyme or when the oxido‐reduction state of the heme is varied. However, a strong effect is exerted by the flavin on the symmetry of the heme environment, this effect being probably an indirect one, via the protein moiety, as discussed in this paper.