| Abstract
| - We have made significant improvements in pump−probetime-resolved X-ray absorption spectroscopy thatenable us to structurally describe chemical intermediates with shortlifetimes. We demonstrate that X-raypreedge data for a 1 mM compound can be acquired with a highsignal-to-noise ratio by time-resolveddiscrimination of fluorescent signals from a 13-element germaniumdetector. With the utilization of thisnovel time-multiplexed laser photolysis system coupled to a flow cell,we characterized the structure of theinitial photoproduct of five-coordinate base-off Co(III)methylcobalamin. The structure of the primaryphotoproduct could have included five- or six-coordinate species withwater ligation, or a four-coordinatesquare-planar species. A four-coordinate Co(II) species isexpected to be unstable but its biological relevanceis highlighted by our recent discovery of a four-coordinate Co(II)species, (existing as the inactive, as isolated,form) in the corrinoid protein of Clostridiumthermoaceticum. The X-ray preedge spectra of five- andsix-coordinate species have a strong 1s−3d transition at about 10 eV belowthe edge. In four-coordinate, square-planar species the 1s−3d intensity is significantly reduced, but theyshow a 1s−4pz peak at about 6 eVbelowthe edge. We used this “fingerprint” to monitor the structuralchange upon photolysis. Since the quantumyield of the base-off species is 0.48, the observed spectrum uponphotolysis is a mixture of photoproduct andinitial states. The photoproduct of the base-off methylcobalaminshows a substantial decrease in the 1s−3dpeak and significant increase in the 1s−4pzpeak. This indicates the formation of a four-coordinatespecies.The four-coordinate species in the free cobalamin is very unstableand can only be detected by time-resolvedmethods. This indicates a special role for the protein inmaintaining an unusual four-coordinate Co(II)corrinoid.
|
