Complex I, the NADH oxidoreductase is the first enzyme in the mitochondrial and bacterial respiratory chain. Eukaryotic Complex I has more than 35 different subunits and a molecular weight of approximately 1 MDa, while the minimal bacterial Complex I has 14 subunits and a molecular weight of approximately 600 kDa. The main role of the respiratory chain is the production of a membrane potential used by the F-ATPase for ATP production. However, the processes in the oxidative phosphorylation system are also involved in many other cellular processes, including the initiation of apoptosis. Defects in Complex I are the cause of many mitochondrial diseases, among them are myopathies, central nervous system disorders, Alzheimer's and Parkinson's diseases. These defects could result in catalytic and/or structural dysfunctions of Complex I. However, its functional principles are little understood and the high resolution structure of Complex I is still unknown, mainly due to its large size and complex assembly. We have visualized for the first time a distinct domain structure of Complex I. This domain structure is conserved in Yarrowia lipolytica and bovine Complex Is, and partially in the bacterial enzyme. In addition, we have been able to localize the possible positions of the catalytic center in Complex I from Y. lipolytica, using the X-ray model of the recently solved structure of a hydrophilic fragment of Complex I from the bacterium Thermus Thermophilus. The localization of the catalytic center has major functional implications, since it favors a conformationally driven active proton pumping mechanism. A coherent model of the structure of Complex I is emerging, however, many questions still need to be addressed. Apparent contradictions within and between current biochemical, biophysical and structural data exist that can only be resolved when different functional states and intermediates of Complex I are structurally analyzed. Vertebrae and fungal Complex Is show a delayed active/deactive state transition in contrast to the bacterial Complex I, which does not. We will analyze these transitions and determine the structural changes in the presence of substrates and inhibitors. The inherent flexibility of Complex I will require a large experimental and computational effort to separately reconstruct the different conformations. This effort will be aided by improvements in our image processing methods and by the creation of an image processing pipeline that will allow the evaluation of the data as they are collected.
Genetic defects of subunits of Complex I are a major cause of mitochondrial diseases. Understanding the still unknown structure and function of this enzyme is crucial for the development of effective treatments. We will determine the 3D structure of Complex I in different catalytic states to obtain insights into the function of this highly complicated enzyme. 1
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