Atypical Features of Thermus thermophilus Succinate:Quinone Reductase

Olga Kolaj-Robin,Frauke Baymann,Mohamed R Noor,Tewfik Soulimane,Sarah R O’Kane,Andreas Hofmann

doi:10.1371/journal.pone.0053559

Abstract

The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His8-SdhB and rcII-SdhB-His6) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the [2Fe-2S] cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.

Highlights

The succinate:quinone oxidoreductases (SQOR) superfamily (EC 1.3.5.1) comprises enzymes serving as the respiratory complex II and are classified depending on the direction of the reaction catalyzed in vivo
As this behavior was not noted to date in other complexes II, to further confirm that the observed cooperativity is a genuine phenomenon and to provide valuable insights into the mechanism of action of a thermophilic succinate:quinone reductase (SQR), a homologous expression system has been developed for this protein
The trimeric forms exhibited cooperativity at high temperatures. This provided the experimental evidence that this novel feature of SQORs reported previously for the native T. thermophilus complex II is a genuine phenomenon of the trimeric enzyme and that each protomer is involved in succinate oxidation

Summary

Introduction

The succinate:quinone oxidoreductases (SQOR) superfamily (EC 1.3.5.1) comprises enzymes serving as the respiratory complex II and are classified depending on the direction of the reaction catalyzed in vivo. While the succinate:quinone reductases (SQRs) mediate the oxidation of succinate to fumarate coupled with the reduction of quinone to quinol, the quinol:fumarate reductases (QFRs) catalyze the reverse reaction [1]. SQR and QFR are homologous proteins that evolved from a common evolutionary ancestor and can catalyze both reactions in vitro and in the cell under the appropriate conditions [2,3]. SQRs are involved in the aerobic metabolism and, as well as being a part of the respiratory chain, constitute the only membrane-bound enzyme of the tricarboxylic acid cycle [4].

Methods

Results

Conclusion