Crosstalk between cytochrome bc1 complex and mitochondrial matrix enzymes

Abstract

Scope and Method of Study:

Protein pull-down assay, western blotting, protein activity assay, superoxide assay, Surface Pasmon Resonance, Mass Spectroscopy, Cross linking.

Findings and Conclusions:

The interactions between the mitochondrial cytochrome bc 1 complex and matrix soluble proteins were studied by a precipitation pull-down technique. One of the matrix proteins pulled down by bc 1 complex was identified as mitochondrial malate dehydrogenase (MDH) by MALDI-TOF Mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using cross linking technique, subunits I, II (core I & II) and V of the bc 1 complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc 1 complex results in the increase of the activities of both the bc 1 complex and MDH. The effect of the bc 1 complex on the activities of MDH is unidirectional (oxalacetate -> malate). These results suggest that the novel crosstalk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics. We also confirmed the existence of a short form of MDH. We found that it does not have the normal enzyme activity but may help the normal long MDH together to regulate the bioenergetics process.

Another matrix protein pulled down by bc 1 complex was identified as mitochondrial aconitase. Subunit II of the bc 1 complex was identified as the interacting site for aconitase. Unlike MDH, no effect on the activity of the bc 1 complex was observed when purified aconitase was interacted with the detergent dispersed bc 1 complex. And the activity of aconitase was only enhanced slightly by cytochrome bc 1 complex. Upon the binding of aconitase, the superoxide generating activity of bc 1 complex is greatly decreased. However, when aconitase was added to the bacterial bc 1 complex, little effect on its superoxide generating activity was observed. The lack of core subunits I and II in the bacterial bc 1 complex may be the reason for this difference. The interaction between bc 1 complex and aconitase can be enhanced by calcium, which is considered as an active factor for the bioenergetic process of mitochondria. These results suggest that the binding between mitochondrial bc 1 complex and aconitase might play an important role in superoxide scavenging when mitochondria is in a high energy producing state, which is crucial for mitochondria and the whole organism.

To study the co-complex of mitochondrial bc 1 complex and malate dehydrogenase (or aconitase), we developed two approaches to obtain the co-crystals. With these approaches, I got some crystals and am conducting further studies.