Establishing proton-pumping stoichiometries with state-of-the art spectroscopy and a new generation of molecular pH probes
- Sindra Peterson Årsköld
A key tool used by nature for energy storage and conversion is the proton gradient. Such a gradient is created when large, membrane-bound enzyme complexes catalyze electron transfer coupled to proton translocation across the membranes of closed vesicles. These endergonic processes are powered by energy from sun-light, in the thylakoid membranes of chloroplasts, or from the oxidation of carbon fuels, in the mitochindrial inner membrane and the bacterial plasma membrane. The pH and charge gradients created, the proton-motive force, is subsequently utilized by ATP synthase to create ATP, the versatile “energy coin” of biology.
Several such proton pumps are known. In the thylakoid membrane, the light reactions of photosynthesis drive proton translocation by both photosystems, as well as the Cyt b6f complex. In the mitochindrial inner membrane, Complex I, the Cyt bc1 complex, and Cyt c Oxidase are known proton pumps. The elucidation of the mechanism of these enzymes - particularly the photosynthetic ones – is important for the development of alternative energy sources. As the respiratory enzymes have been shown to play a part in degenerative diseases such as Parkinson’s disease, understanding of these proton pumps has potential medical relevance.
In order to understand the pumping mechanism of these enzymes, it is essential to establish the stoichiometry of protons pumped per electron transferred through each enzyme. The number of electrons transferred can be controlled by the amount of reductant/oxidant added to a sample. Monitoring the number of protons translocated, however, is less straight-forward. Several methods to measure the stoichiometries have been employed, ranging from the use of conventional pH electrodes, combinations of membrane permeable and non-permeable pH-sensitive dyes or calculating the equilibrium thermodynamic force ratio. Whereas these methods have yielded important results, these results are not always unambiguous. This is due to technical and practical flaws and difficulties associated with the methods.
We are developing a novel, robust and generally applicable technique for proton-pumping measurements in biological membrane vesicles, with the aim of elucidating the coupling mechanism in bioenergetic enzymes, particularly respiratory Complex I and the photosynthetic Cyt b6f complex.