Polarized proton spin density images the tyrosyl radical locations in bovine liver catalase

What is it about?

Promising the title 'Polarized proton spin density images the tyrosyl radical locations in bovine liver catalase' sounds, an explanation seems to be appropriate. From NMR spectroscopy it is well established that protons near to an unpaired electron of a radical, differ from those of the bulk in many respects. This is true both for the relaxation of the proton spins, and their polarisation by a method called dynamic nuclear polarisation (DNP). The close protons in contact with the contact with the non-Zeeman electron spin - electron spin interaction reservoir mediate both relaxation and polarisation of the bulk protons. They close protons are not numerous. Typically there may be twenty close protons inside a magnetic nuclear spin diffusion barrier surrounding an unpaired electron, which is less than 1 out of 100 protons in a radical protein like tyrosyl doped catalase. Polarized spins, nuclear or electronic, are 'seen' by polarized neutrons. The first thing to do is to have a look at the unpaired electrons. A reasonably high polarisation of unpaired electrons is achieved at liquid helium temperatures in a moderately strong magnetic field of several Tesla (also a prerequisite for DNP). A small contribution of electron polarisation dependent (or magnetic) neutron scattering to the total intensity of neutron scattering from the radical protein in solution would be prohibitively difficult to measure. Polarized nuclear spins, and proton spins in particular, are more rewarding targets of polarized neutron scattering. First their polarisation dependent scattering length is five times larger than that of electrons, and second, the number of close protons per radical polarized by DNP may be larger than 1. In the case of tyrosyl doped catalase, the polarisation of the 20 close protons changed by 0.1 by inverting the direction of DNP resulting in 2 protons polarized in excess. This is one of the marvellous things about DNP: you can change the direction from positive to negative proton polarisation just by switching from a microwave frequency slightly below the electron paramagnetic resonance (EPR) to a frequency slightly above the EPR. The gradient of proton polarisation which develops inside the magnetic proton spin diffusion barrier at the onset of DNP is short-lived. Therefore the direction of DNP is changed after a few (typically 5) seconds and during each period of 5s the intensity of polarised neutron scattering is recorded by an area detector a hundred times. A good accuracy of the neutron scattering data in each of its time slots of 50 ms is obtained after several thousand cycles. The time dependent intensity of polarised neutron scattering intensity reflects the build up of proton polarisation near the radical sites in space and time, whence the name TPP of the method , which stands for time-resolved proton polarisation. The simultaneous measurement of the proton NMR intensity at a more modest time resolution of 1s supports TPP from the bulk protons. The analysis of the time resolved data of neutron small-angle scattering data and of NMR shows that 4 of the 20 tyrosine amino acids of each of the four subunits of catalase might have been converted to a radical state. They are all close to the centre of the catalase molecule. Among these is tyr-369 which has been identified as a tyrosyl by EPR. TPP using small-angle scattering provides a radial probability distribution of radical sites in proteins, at present. An angular resolution in terms of polar angles seems to be possible with more accurate data. Further improvements concern the choice of frequency and power of microwave irradiation allowing for a more efficient DNP, the use of an area detector which can cope with higher neutron scattering intensities, and a better control of the time frame generator, just to mention some points. A leaking out of the neutron beam intensity can be avoided by putting the neutron spin polarizer just behind the velocity selector, for instance.

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