期刊
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
卷 1807, 期 1, 页码 68-79出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbabio.2010.10.009
关键词
Complex II; Succinate:quinone oxidoreductase; Succinate dehydrogenase; Cooperativity; Thermus thermophilus
资金
- Science Fundation Ireland [BICF685]
- French National Centre for Scientific Research (CNRS)
- SFI Ulysses
Enzymes serving as respiratory complex II belong to the succinate:quinone oxidoreductases superfamily that comprises succinate:quinone reductases (SQRs) and quinol:fumarate reductases. The SQR from the extreme thermophile Thermus thermophilus has been isolated, identified and purified to homogeneity. It consists of four polypeptides with apparent molecular masses of 64, 27, 14 and 15 kDa, corresponding to SdhA (flavoprotein), SdhB (iron-sulfur protein), SdhC and SdhD (membrane anchor proteins), respectively. The existence of [2Fe-2S], [4Fe-4S] and [3Fe-4S] iron-sulfur clusters within the purified protein was confirmed by electron paramagnetic resonance spectroscopy which also revealed a previously unnoticed influence of the substrate on the signal corresponding to the [2Fe-2S] cluster. The enzyme contains two heme b cofactors of reduction midpoint potentials of -20 mV and -160 mV for b(H) and b(L), respectively. Circular dichroism and blue-native polyacrylamide gel electrophoresis revealed that the enzyme forms a trimer with a predominantly helical fold. The optimum temperature for succinate dehydrogenase activity is 70 degrees C, which is in agreement with the optimum growth temperature of T. thermophilus. Inhibition studies confirmed sensitivity of the enzyme to the classical inhibitors of the active site, as there are sodium malonate, sodium diethyl oxaloacetate and 3-nitropropionic acid. Activity measurements in the presence of the semiquinone analog, nonyl-4-hydroxyquinoline-N-oxide (NQNO) showed that the membrane part of the enzyme is functionally connected to the active site. Steady-state kinetic measurements showed that the enzyme displays standard Michaelis-Menten kinetics at a low temperature (30 degrees C) with a K-M for succinate of 0.21 mM but exhibits deviation from it at a higher temperature (70 degrees C). This is the first example of complex II with such a kinetic behavior suggesting positive cooperativity with k' of 0.39 mM and Hill coefficient of 2.105. While the crystal structures of several SQORs are already available, no crystal structure of type A SQOR has been elucidated to date. Here we present for the first time a detailed biophysical and biochemical study of type A SQOR a significant step towards understanding its structure-function relationship. (C) 2010 Elsevier B.V. All rights reserved.
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