Abstract's Details

Photoreduction of Metalloprotein Active Sites by Synchrotron Radiation
Abstract ID	BIO-04
Presenter	Matthew Latimer
Presentation Type	Poster
Full Author List	M.J. Latimer (1), M.C. Corbett (2), T.L. Poulos (3,4), I.F. Sevrioukova (3), K.O. Hodgson (1,2), B. Hedman (1)
Affiliations	(1) SSRL (2) Department of Chemistry, Stanford University (3) Departments of Molecular Biology & Biochemistry, UC Irvine (4) Physiology & Biophysics, and Chemistry, UC Irvine
Category	Bio/Life Sciences
Abstract	X-ray damage to protein crystals is often assessed on the basis of the degradation of diffraction intensity, yet this measure is not sensitive to the rapid changes that occur at photosensitive groups, such as the active sites of metalloproteins. In studies to be presented, x-ray absorption spectroscopy (XAS) is used to study the x-ray dose-dependent photoreduction of crystals of the [Fe(2)S(2)]-containing metalloprotein, putidaredoxin. A dramatic decrease in the rate of photoreduction, followed through changes in the XAS edge structure, is observed in crystals cryocooled with liquid helium at 40 K as compared to those cooled with liquid nitrogen at 110 K. Whereas structural changes consistent with cluster reduction occur in the active site of the crystal measured at 110 K, no such changes occur in the crystal measured at 40 K, even after an eight-fold increase in dose. When the structural results from extended x-ray absorption fine structure measurements (EXAFS) are compared to those obtained by crystallography on this and similar proteins, it is apparent that x-ray induced photoreduction has impacted the crystallographic data and subsequent structure solutions. These results strongly indicate the importance of using liquid helium-based cooling for metalloprotein crystallography in order to avoid the subtle, yet important changes that can take place at the metalloprotein active sites when liquid nitrogen-based cooling is used. The study also illustrates the need for direct measurement of redox states of the metals, through x-ray absorption spectroscopy, simultaneously with the crystallographic measurements.
Footnotes
Funding Acknowledgement	The work was performed at Stanford Synchrotron Radiation Laboratory (SSRL) with support from the NIH NCRR BTP program and the US DOE BER. SSRL operations are funded by the US DOE BES.