High-Field Electron Paramagnetic Resonance Spectroscopy of Whole Organisms – SeediEPR

We are able to get our results by using very high-magnetic fields (10 T or 50000 times earth’s magnetic field) and very high frequency microwaves (95 to 300 GHz compared 1 GHz for portable telephones). Together they allow us to obtain very detailed information about the manganese in proteins and cells. There are very few laboratories in the world that are able to make such measurements.

We have determined the in situ chemical distribution of manganese(II) in intact Deinococcus radiodurans. This is only the second time this has ever been done on a whole organism. However unlike the first, our measurements were sufficiently resolved to detect Mn(II) bound to proteins. These measurements will allow us to understand the function of certain manganese species and how maganese is acquired and transported in cells. This is important since manganese is implicated in many important biological processes, such as oxygen production by photosystem 2 in plant cells and protection against reactive chemicals. These preliminary measurements show us that the same type of measurements can be made on seeds.

The project has been going on for less than 6 months. Therefore, it is very difficult to appreciate what its impact will be.

A paper on our initial results which identifies the manganese species in Deinococcus radiodurans and how they change as a function time has been submitted to the Journal of the American Chemical Society.

There are only a limited number of spectroscopic techniques capable of monitoring biochemical processes involving metal ions and radicals in whole organisms. This proposal addresses the potential of using high magnetic-field high microwave-frequency electron paramagnetic resonance (HFEPR) techniques exactly for these purposes. The system on which we shall first focus is the seed from the plant model Arabidopsis thaliana, in particular, its manganese homeostasis and radical chemistry. Manganese plays a crucial role in a number of important processes including photosynthetic oxygen production and control of oxidative stress while the radicals present in the seeds reflect reactive oxidative chemistry. EPR is naturally suited to study these two very different paramagetic species. It has already been established that HFEPR is vastly superior over conventional 9 GHz EPR for studying radicals and Mn(II) ions, the predominant form of manganese in organisms. Preliminary measurement on Arabidopsis seeds have shown that well-defined HFEPR signals of high quality can be detected from both types of paramagnetic centers. The primary goal of this project is to develop techniques to understand in detail the identity and electronic structure of the centers that contribute to the HFEPR spectra. To do this, a comparative approach will be taken and a database of small Mn(II) complexes will be developed. For radicals, we have already such a library based on previous work. This comparative approach will be augmented by the use of 95 GHz high magnetic-field electron nuclear double resonance (HFENDOR), a method that provides an NMR map of nuclear spins (e.g. proton, phosphorous, nitrogen atoms) near the paramagnetic centers. In the case of Mn(II), this will give a detailed structure of the ligand sphere around the metal centers and for radicals it will provide information about the identity of the radicals and their interactions with their environment (e.g. hydrogen bonds). Once an understanding of wild-type Arabidopsis seeds has been established, this knowledge will be used to study how Mn(II) speciation varies in other seeds and changes as a function of physiological effects, such as hydration/imbibation. The effect of metal transport proteins on manganese speciation will also be examined, in particular the nramp proteins that control the motion of Mn(II) out of plant vacuoles. For the radicals, the effect of various mutations of the biosynthetic pathways of quinones, fatty acids and flavonoids will be studied to identify the radicals and their role in oxidative damage and control. Finally, these measurements will be extended to other organisms notably: (1.) Saccharomyces cerevisiae for which a similar approach has been recently taken, but at much lower frequencies and (2.) Deinococcus radiodurans which has been shown to have an exceptional capacity for concentrating large amounts of vacuolar Mn(II) (> 30 mM). The approaches described will be the first attempted at such high frequencies and far more in-depth than previous studies at lower frequencies. They will define how high-field EPR techniques can be used in the study of whole organisms.