From Time-Averaged to Time-Resolved Crystallography: Studies on Superoxide Dismutase and Myoglobin

We have studied protein motions with nanosecond time-resolved crystallography. The release of carbon monoxide from its complex with myoglobin (MbCO) was triggered by nanosecond laser pulses. X-ray data were collected with time delays ranging from 4 ns to 1.9 ms using the Laue method, at the European Synchrotron Radiation Facility in Grenoble. For the first time point, X-ray pulses of 150 ps duration were used. Electron difference maps show the release of the CO and the subsequent motion of the iron out of the haem plane. In the 4 ns difference map, a positive density feature is found at a position coinciding with that of an intermediate docking site seen at low temperature. The following time points show the rebinding of the CO, and also indications of protein relaxations extending further than 4 ns. This is the first time-resolved protein crystallography experiment with nanosecond time-resolution yielding structural results.

We have developed instrumentation and methods for time-resolved protein crystallography, including a method to reduce the noise in electron density difference maps from poorly accurate data. The method is also applicable to other cases where the signal to noise ratio is low.

The crystal structure of iron superoxide dismutase (SOD) from the hyperthermophilic archaeon <i>Sulfolobus solfataricus</i> has been determined to 2.3 Å resolution by molecular replacement. The structure revealed an increased number of inter-subunit ion-pairs in a compact tetramer. We suggest this to be important for the thermostability. However, the general fold is found to be similar to other known iron- or manganese SOD structures. In addition, the electron density maps revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site.