Photoreduction of the active site of the metalloprotein putidaredoxin by synchrotron radiation.
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. Here, X-ray absorption spectroscopy 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 is observed in crystals cryocooled with liquid helium at 40 K compared with 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 eightfold increase in dose. When the structural results from extended X-ray absorption fine-structure measurements are compared with those obtained by crystallography on this and similar proteins, it is apparent that X-ray-induced photoreduction has had an impact on 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 the redox states of the metals, through X-ray absorption spectroscopy, simultaneously with the crystallographic measurements.
Corbett
Mary C
MC
Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Latimer
Matthew J
MJ
Poulos
Thomas L
TL
Sevrioukova
Irina F
IF
Hodgson
Keith O
KO
Hedman
Britt
B
D001665:Binding Sites; D018360:Crystallography, X-Ray; D005288:Ferredoxins / Q000737:chemistry*; D006371:Helium; D008667:Metalloproteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D010777:Photochemistry; D011506:Proteins / Q000737:chemistry*; D014965:X-Rays / Q000009:adverse effects
D005288:Ferredoxins; D008667:Metalloproteins; D011506:Proteins; D006371:Helium; C100137:putidaredoxin