Photoreduction of the active site of the metalloprotein putidaredoxin by synchrotron radiation

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. 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.

Origin and temperature dependence of radiation damage in biological samples at cryogenic temperatures

“…The occupancy decay at 50 K is reduced by a factor of about 4 compared to 100 K. Cooling down to 5 K did not yield any further improvements. This observation follows the trend reported in recently published XANES data (25,27), where photoreduction of metal B A Fig. 2.…”

“…larger effect was found in x-ray absorption spectroscopy studies addressing specific damage to the metal sites in metalloproteins (25,26). Photoreduction of protein-bound metal clusters was significantly slowed at temperatures below 100 K (25,27). However, photoreduction of metal clusters occurs at doses which are one to two orders of magnitude lower than the dose limit for global damage of around 30 MGy in cryocrystallography at 100 K (28,29).…”

Radiation damage in protein crystals examined under various conditions by different methods

“…20 K), thereby demonstrating an apparent decrease in radiation damage at these lower temperatures. There is evidence from both X-ray diffraction and EXAFS (Yano et less radiation damage between 7 and 40 K compared with 100 K. In principle, a metal atom could be reduced with minimum movement of the surrounding atoms and it might not be expected that additional protection would be conferred at 40 K compared with 100 K. However, significant protection (a factor of 30) has been observed as monitored by XANES measurements (Corbett et al, 2007). The explanation given by Corbett et al (2007) is that 'the electrons generated by X-ray radiolysis are randomly distributed with respect to a metal site and therefore only a small subset would likely be optimized for an athermal reaction'.…”

“…Many of these changes can be observed by a variety of spectroscopic techniques [e.g. UV/Vis spectroscopy (Beitlich et al, 2007;McGeehan et al, 2009), Raman (Carpentier et al, 2007), XAS (Corbett et al, 2007) and EPR (Utschig et al, 2008)]. When trapped in water, the hydrated (or more generally solvated) electrons give a characteristic broad optical absorption spectrum (Ershov & Pikaev, 1968).…”

Future challenges for x-ray microscopy

“…Additional effects include the evolution of hydrogen gas caused in large part by irradiation of water [13]; it may be that temperature-dependent differences in hydrogen gas diffusion through ice matrices help explain why cryo electron microscopy of hydrated organic materials shows less bubbling [14] at temperatures of 40-110 K than at below 15 K [15,16]. The situation where temperatures below 15 K seem more important is for the minizatoin of radiation damage at metal centers in organic molecules [17].…”