Study of the Hansenula anomala yeast flavocytochrome‐b2‐cytochrome‐c complex

Abstract

The reversible association of the Zn²⁺ ‐substituted Hansenula anomala cytochrome c dimer (Thomas et al., preceding paper in this issue) to flavocytochrome b₂ in oxidized or lactate‐reduced state has been investigated by fluorimetry. The same method has been used for the determination of Zn‐cytochrome c complexing to defined proteolytic fragments of flavocytochrome b₂, either heme‐b₂‐containing monomers or a flavin‐linked tetramer. All these fragments but the isolated cytochrome b₂ core showed binding stoichiometries, K_d values and ionic strength dependences quite similar to those found for native flavocytochrome b₂. These data allowed localization of the single high‐affinity binding site of cytochrome c on a particular globule in the dehydrogenase domain of the flavocytochrome b₂ protomers. Quenching of the Zn‐porphyrin c fluorescence in the various complexes occurred with only minor changes of the fluorescence lifetime and did not show any direct relationship to the presence or the redox state of the heme b₂ group.