Structural Studies of Yeast Flavocytochrome b2: Cooperative Roles of the alpha and beta Globules in the Formation of the Flavin-Binding Sites

Abstract

The purpose of the study reported here was the localization of the heme binding sites on the two globular fragments, α and β, of the ‘cleaved’ form of the flavocytochrome b2 chain. These fragments were partially resolved by means of molecular sieving under denaturing conditions (3 M or 6 M guanidine in the presence of 2‐mercaptoethanol). They were then renatured in the presence of excesses of FMN and protoheme. The protoheme was found to be quantitatively bound to the α subunit, confirming previous findings. The flavin binds neither to α alone nor to β alone, but only to the reassociated αβ protomer. The results are discussed in terms of the possible occurrence of gene fusion in the formation of the complex flavocytochrome chain of this very particular L‐lactate cytochrome c reductase found specifically in yeasts.

Study of a Zone Highly Sensitive to Proteases in Flavocytochrome b2 from Saccharomyces cerevisiae

“…In multifunctional proteins, a zone of preferential proteolysis is usually called a hinge or fringe region [37]. But its existence proves the occurrence of domains only when these can be separated under non-denaturing conditions [1,2], which is not the case for flavocytochrome h2 [14]. Thus, the possibility that the flavodehydrogenase unit consists of two domains built up by continuous segments (namely fragment j on one hand and the C-terminal two thirds of fragment x. on the other) is supported but not proven by our proteolysis experiments.…”

“…On sodium dodecylsulphate/acrylamide gel elec- trophoresis, the nicked enzyme shows two bands of molecular weight around 33000-36000 (fragment uj and 21 000 (fragment fl). Prior to denaturation, the nicked enzyme is still a tetramer, with somewhat modified properties compared to the native enzyme [3,7,13] and the fragments can only be separated under denaturing conditions [14]. With subtilisin and trypsin, further degradation takes place, eventually leading to inactive fragments [7,15].…”

A Flavin‐Mononucleotide‐Binding Site in Hansenula anomala Nicked Flavocytochrome b2, Requiring the Association of Two Domains

“…This procedure leads [14] to renaturation and maximal flavin binding to functional flavin sites before flavin has been extensively diluted out of the bag. To this end, protein concentrations have to be in large excess over the dissociation constant of the flavoprotein.…”

“…As far as polyglobular proteins are concerned, one problem was to find out Two Domains for F M N Site in Flavocytochrome hz whether or not the folding of each globular domain required interactions with the adjacent domains before the final phase in which both domains come into close contact [9, lo]. Another problem was to understand how the association of several different subunits or domains allowed the formation of functional sites which are undetectable in any of the isolated domains (for example for coenzyme binding [14,39]). …”

“…Bottom: the triglobular model has been proposed for the H . anomah enzyme [5,11] taking into consideration (a) the degradation scheme (top), (b) the data on the explosion of the molecule after cleavage of the loops 'cd', the four cytochrome cores being detached from an flavodehydrogenase entity, and (c) the location of the flavin site at the zfl interface as proposed for the S. cerevisiue enzyme [14]. This model finds additional support (a) in the present study that confirms, for the H .…”