Hi James,

We used to talk about primary and secondary radiation damage. The former 
operates at room temperature where free radicals were said to be formed 
in solution and diffuse around to damage proteins. Under cryoconditions 
this no longer happens, leading to greatly improved crystal life time, 
but we still have primary radiation damage, with the photons directly 
hitting the protein.

It was my understanding that this was still considered to form a free 
radical at the affected atom without there being any diffusion involved. 
Sulfur atoms would be more sensitive as they have a larger X-ray 
cross-section or because they may act as free-radical sinks where free 
radicals generated nearby strip an electron from the sulfur, thereby 
satisfying their own electronic configuration and converting the sulfur 
into a radical state. For instance, in ribonucleotide reductase a 
tyrosine free radical is formed spontaneously (using oxygen and an 
dinuclear iron site) and "jumps" over 20 Angstrom from one subunit to 
another to form a thiyl free radical in the active site. It then "jumps" 
back to the tyrosine upon completion of the catalytic cycle. Although we 
don't know how it jumps, certainly not by diffusion, there is general 
agreement that it does happen.

People have also observed broken disulfides in cryocrystal structures 
with the sulfurs at a distance that is too long for a disulfide but too 
short for a normal non-bonded sulfur-sulfur interaction. I seem to 
remember that this distance was suggested to indicate the presence of a 
thiyl free radical. I'm no chemist of physicist so can't evaluate if 
that claim is reasonable but if it is then that would be direct evidence 
to support the involvement of a free radical state in radiation damage.

So I guess my questions/comments are
- what are the great many good reasons to think that free radicals do 
NOT play a role in radiation damage under cryo.
- although diffusion does not happen below 130K, radicals do appear to 
teleport, at least over short distances.

Bart

James Holton wrote:
> I don't mean to single anyone out, but the assignment of "free radicals" 
> as the species mediating radiation damage at cryo temperatures is a "pet 
> peeve" of mine.  Free radicals have been shown to mediate damage at room 
> temperature (and there is a VERY large body of literature on this), but 
> there are a great many good reasons to think that free radicals do NOT 
> play a role in radiation damage under cryo.
> 
> This "assignment" of free radicals to damage is often made (flippantly) 
> in the literature, but I feel a strong need to point out that there is 
> NO EVIDENCE of a free radical diffusion mechanism for radiation damage 
> below ~130K.  To the contrary there is a great deal of evidence that 
> water, buffers and protein crystals below ~130 K are in a state of 
> matter known as a "solid", and molecules (such as free radicals) do not 
> diffuse through solids (except on geological timescales).  If you are 
> worried that the x-ray beam is heating your crystal to >130 K, then have 
> a look at Snell et. al. JSR 14 109-15 (2007).  They showed quite 
> convincingly that this just can't happen for anything but the most 
> exotic situations.
> 
> There is evidence, however, of energy transfer taking place between 
> different regions of the crystal, but energy transfer does not require 
> molecular diffusion or any other kind of mass transport.  In fact, 
> solid-state chemistry is generally mediated by cascading 
> neighbor-to-neighbor reactions that do not involve "diffusion" in the 
> traditional sense.  Electricity is an example of this kind of chemistry, 
> and these reactions are a LOT faster than diffusion.  The closest 
> analogy to "diffusion" is that the propagating reaction can be seen as a 
> "species" of sorts that is moving around inside the sample.  Entities 
> like this are formally called quasiparticles.  Some quasiparticles are 
> charged, but others are not.  If you don't know what a quasiparticle is, 
> you can look them up in wikipedia.
> Some have tried to rescue the "free radical" statements about radiation 
> damage by claiming that individual electrons are "radicals".  I guess 
> this must come from the "pressure" of such a large body of free-radical 
> literature at room temperature.  However, IMHO this is about as useful 
> as declaring that every chemical reaction is a "free radical" reaction 
> (since they involve the movement of electrons).   I think it best that 
> we try to call the chemistry what it is and try to stamp out rumors that 
> mechanisms are known when in reality they are not.
> 
> Just my little rant.
> 
> -James Holton
> MAD Scientist
> 
> 


-- 

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Bart Hazes (Assistant Professor)
Dept. of Medical Microbiology & Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta
Canada, T6G 2H7
phone:  1-780-492-0042
fax:    1-780-492-7521

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