Linking proton pumping to hydride transfer: mechanism of transhydrogenase

University Of Illinois At Urbana-Champaign, Urbana IL

Investigators

Abstract

One essential feature of all life is the ability to extract energy from the environment and convert it to useful work. Humans, other animals, plants and microbes all share a set of strategies for conserving and converting energy from one form to another. A central element in these strategies is to generate and then utilize an electrical voltage across a membrane. Energy in one form is used to separate positive and negative charges across a membrane, and then this voltage is used to drive a current of ions that is used to do work such as make ATP, expel toxins or concentrate useful molecules. Membrane spanning proteins have evolved to both generate and utilize the voltage across the membrane. Since protons are the most common ions that are transported across the membrane for these purposes, the measurement of the capacity for work is called the proton motive force. This project is focused on one protein called the transhydrogenase that uses the proton motive force to drive a chemical reaction to form a molecule, NADPH, that is essential for many cellular processes. For example, NADPH is needed for commercial microbial production of fermentation products, and manipulating the amount of transhydrogenase in the organism is one way to increase product yield. The project is aimed at determining the way in which a flux of protons flowing through the transhydrogenase protein from one side of the membrane (electrically positive) to the other (electrically negative) is coupled to driving a chemical reaction at a different location on the protein. The project will involve students at all levels; undergraduates, graduate students, including two women, and postdocs. The highly interdisciplinary scope of the research will allow students to be trained in a broad range of biochemical and biophysical techniques. This project is devoted to using biochemical and biophysical methods to examine the mechanism by which the membrane transhydrogenase couples hydride transfer from NADH to NADP+ to the proton electrochemical gradient across the membrane. The PI and his collaborators have determined the high-resolution structure of the protein that revealed that one domain of the 3-domain protein can occupy two dramatically different spatial orientations, more or less flipped by 180o. Based on this structure it can be postulated that in one configuration the substrate binds in a way that opens a proton channel through the membrane from one side only, allowing a histidine buried within the membrane to be protonated. The domain then "flips" and in the second configuration the product is formed by hydride transfer. The domain then flips back to the initial configuration in which the bound product induces the proton channel to open in the opposite direction, allowing proton dissociation. The model assumes large conformational changes during the catalytic cycle and also proposes a linkage between substrate binding and proton binding. These hypotheses will be experimentally tested using a combination of molecular biology, biochemistry and biophysical methodologies. NADPH binding to the enzyme will be tested using fluorescence anisotropy measurements. The enzyme will be reconstituted in proteoliposomes to allow the adjustment of the internal and external pH values along with the transmembrane voltage. Conformational dynamics will be examined by using distance-dependent spectroscopic methods using pairs of spin probes (PELDOR) and fluorescent probes (FRET) using modified cysteines placed at various regions in the protein.