Characterization of Fumarate Reductase from Baker's Yeast: Essential Sulfhydryl Group for Binding of FAD

Abstract

Fumarate reductase apoenzyme having the ability to reconstitute active enzyme was obtained by dialyzing the holoenzyme against 1 M KBr. The dissociation constant of the FAD-apoenzyme complex was 2.3 X 10(-8) M. The denatured holoenzyme and apoenzyme possessed seven sulfhydryl (SH) groups as determined with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In the native apoenzyme, five SH-groups reacted with DTNB, and four of them were completely protected by the addition of FAD, while in the native holoenzyme, one was modified without inactivation. These results indicate that one SH-group is located on the surface of the enzyme molecule, four at or near the FAD-binding site, and two deeply embedded in the molecule. The modification of the apoenzyme caused inhibition of binding of FAD, resulting in loss of the ability to reconstitute enzymatic activity. Analyses of the data by statistical and kinetic methods suggested that a reactive SH-group is involved among the four SH-groups in the binding of FAD to the apoenzyme.

Function and Structure of Complex II of the Respiratory Chain

“…It had been predicted, based upon nucleotide and amino acid sequence comparison of QFR and SQR as well as biochemical analysis of their proposed structures, that they have a common evolutionary precursor (32)(33)(34)(35)(36). A class of soluble fumarate reductases is found in many anaerobic or microaerophilic bacteria such as from the genus Shewanella (37), yeast (38), and unicellular parasites like Trypanosoma brucei (39). Although these enzymes do not exhibit many of the properties of the classical complex II, the flavoprotein domain is homologous to the flavoprotein subunit of SDH and FRD.…”

“…Although these enzymes do not exhibit many of the properties of the classical complex II, the flavoprotein domain is homologous to the flavoprotein subunit of SDH and FRD. Two characteristics of this soluble class of FRD differ from classical complex II: (a) the flavin is noncovalently bound to the enzyme; and (b) the enzymes are essentially unable to oxidize succinate (37)(38)(39)(40). Similar to what is found in nature, when sitedirected mutants of E. coli QFR (41) or Saccharomyces cerevisiae SQR (42) were constructed that produce enzymes containing noncovalently bound flavin, the enzymes had lost the ability to oxidize succinate, although they retained the ability to reduce fumarate.…”

“…All SQR and QFR complexes from both prokaryotes and eukaryotes that have been examined in detail contain a histidyl-FAD covalent linkage. By contrast, soluble fumarate reductase homologs of the flavoprotein subunit of complex II such as those from yeast (38), bacteria from the genus Shewanella for example (37), and unicellular parasites (39) contain noncovalently bound FAD. It has been speculated that the covalent linkage prevents the loss of the flavin cofactor from proteins in a membrane or periplasmic environment where concentrations of flavin mononucleotide (FMN) and FAD would be low and unable to replace the lost flavin (81).…”

A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens

“…The enzyme consists of a single polypeptide chain (64 kDa), the sequence of which resembles those of the catalytic subunits of the membranous fumarate reductases. A soluble fumarate reductase containing FAD has also been isolated from Saccharomyces cerevisiae (Muratsubaki and Katsume 1985). Using an antiserum raised against fumarate reductase from S. putrefaciens, we attempted to determine whether W. succinogenes forms a related protein.…”

Purification and characterization of fumarate reductase from Methanobacterium thermoautotrophicum

“…On the contrary, fumarate reductases isolated from brewers yeast (65) and bakers yeast (49) were found to be in the cytosol fraction of the Reducing agents helped in protecting against loss in the enzyme activity, provided that a strict anaerobic environment was maintained. Furthermore, unlike the results Nozaki et al (66) observed, the inactivated enzyme could not be reactivated by incubation with reducing agents under anaerobic conditions.…”