Ultrafast X-Ray and Optical Spectroscopies on Vitamin B12: Dynamics and Mechanistic Insight

The photochemistry of vitamin B12 derivatives have been further investigated by ultraviolet-visible and hard x-ray spectroscopies. Vitamin B12 is essential for the function of many enzymes and a class of photosensing proteins. The lowest energy excited states of B12 are a function of the axial ligand. The scientific research in this dissertation demonstrates further complexity of the ligand-photochemistry relationship. A category of synthetic vitamin B12-inibiting compounds called antivitamins B12 were studied with time-resolved spectroscopy. These antivitamins define new S1 excited states of B12 and establish their own behaviors on the cobalamin spectrum. The acetylidecobalamins show deactivation on a ca. 70 ps timescale from a ligand-field region of the S1 potential energy surface. These acetylidecobalamins are a robust, photostable antivitamin B12. The arylcobalamins demonstrate 90 percent deactivation on a ca. 200 ps timescale. The remaining 10 percent form a long-lived base-off, water-on cobalamin. This is the first unequivocal observation of a ligand-exchange reaction from the vitamin B12 excited state. Comparison of the arylcobalamin and adenosylcobalamin demonstrates no clear relationship between the S1 state and the photochemical reaction yield. Ultrafast X-ray spectroscopy on cyanocobalamin provides a basis for understanding the excited state relaxation on highly-reactive potential energy surfaces. Ultrafast optical-pump, X-ray-probe experiments measure the femto-picosecond bond elongation of cyanocobalamin and subsequent relaxation to the S1 minimum. These measurements support a ballistic relaxation from the Franck-Condon region toward the S1 minimum. Simulations of the measurements show large bond elongation of ca. 20 percent at the S1 minimum. These are also the first polarized femtosecond X-ray absorbance experiments, allowing the separation of anisotropic contributions to the X-ray absorbance from an isotropic sample. These measurements allow a near-quantitative decomposition of the X-ray spectra into molecule-fixed axes. We demonstrate the extreme elongation of the axial bonds, with modest changes of the corrin-metal bond lengths. The final chapters of this dissertation address the application of microdispensing samples for time-resolved x-ray spectroscopy. In chapter 4, we demonstrate the use of a microdispenser to deliver precise picoliter sample volumes for each laser shot. While this sample delivery method provides noisier data than the conventional liquid jet, we demonstrate methods to reject bad microdrop data and correct for positional deviation of drops. Overall, the experiment provides sufficient signal-to-noise data for meaningful interpretation. The acetylide cobalamin undergoes less bond elongation than the cyanocobalamin case. The S1 of the acetylide cobalamin can be assigned to a distribution of axial bonds lengths for the excited state. Finally, in chapter 5 we performed time-resolved spectroscopy on a photosensing protein, CarH. Our measurements partially agree with the femto-nanosecond spectroscopy in the literature. The CarH protein forms a nanosecond-lived MLCT excited state. This is the longest-lived excited state measured for any cobalamin. While the MLCT state resembles the S1 state of methylcobalamin, the reaction rates are quite different. The methylcobalamin case exhibits clear bond homolysis with a rate ca. 1 ns^-1. The CarH demonstrates no clear photoreaction on the timescale of our measurements. Overall, these data demonstrate that the S1 state minimum of a given cobalamin is not deterministic of the photochemical reaction rate. Preliminary X-ray data of the CarH was collected via the microdispenser method. The combined chapter 4 and 5 data from microdispensers demonstrate the feasibility of femtosecond X-ray spectroscopy on precious synthetic and biological samples.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/151755/1/millena_1.pd