Probing the Excited State of Methylcobalamin Using Polarized Time-Resolved X-ray Absorption Spectroscopy

Abstract

We use picosecond time-resolved polarized Xray absorption near-edge structure (XANES) measurements to probe the structure of the long-lived photoexcited state of methylcobalamin (MeCbl) and the cob(II)alamin photoproduct formed following photoexcitation of adenosylcobalamin (AdoCbl, coenzyme B 12 ). For MeCbl, we used 520 nm excitation and a time delay of 100 ps to avoid the formation of cob(II)alamin. We find only small spectral changes in the equatorial and axial directions, which we interpret as arising from small (<∼0.05 Å) changes in both the equatorial and axial distances. This confirms expectations based on prior UV−visible transient absorption measurements and theoretical simulations. We do not find evidence for the significant elongation of the Co−C bond reported by Subramanian et al. [J. Phys. Chem. Lett. 2018, 9, 1542−1546 following 400 nm excitation. For AdoCbl, we resolve the difference XANES contributions along three unique molecular axes by exciting with both 540 and 365 nm light, demonstrating that the spectral changes are predominantly polarized along the axial direction, consistent with the loss of axial ligation. These data suggest that the microsecond "recombination product" identified by Subramanian et al. is actually the cob(II)alamin photoproduct that is produced following bond homolysis of MeCbl with 400 nm excitation. Our results highlight the pronounced advantage of using polarization-selective transient X-ray absorption for isolating structural dynamics in systems undergoing atomic displacements that are strongly correlated to the exciting optical polarization.

Antivitamins B12 in a Microdrop: The Excited-State Structure of a Precious Sample Using Transient Polarized X-ray Absorption Near-Edge Structure

“…Previous work on the ultrafast and picosecond XANES of other B 12 compounds has given a cobalt-eye view of cobalamin bonding, − and polarized XANES difference spectra allow examination of structural changes in a molecule-fixed framework, further refining the structure of the excited state. − Ultrafast measurements on cyanocobalamin (CNCbl) demonstrated a ballistic, slightly underdamped expansion of the axial cobalt–ligand bonds . The structure of this excited state is in good agreement with recent TD-DFT calculations. − In contrast, our study of methylcobalamin (MeCbl) demonstrates that the excited state for this molecule is dominated by changes in the corrin ring rather than changes in the axial bonds . The combination of theoretical calculations of the potential energy surfaces (PESs), transient optical spectroscopy, and ultrafast X-ray spectroscopy using state-of-the-art X-ray free electron lasers (XFELs) allows us to converge on a “molecular-snapshot” view of bond evolution in the excited state.…”

“…For F 2 PhEtyCbl, the ratio of Δ I y + z to ΔI x is ∼4.5:1. This is significantly smaller than that for CNCbl (∼6.5:1) where axial bond elongation dominates , but larger than that for MeCbl (∼2:1) where axial and equatorial changes are comparable . These comparisons indicate that the combined axial expansion is larger than the ring expansion in F 2 PhEtyCbl, although it is smaller than the axial expansion in CNCbl.…”

“…Decomposition of the XANES difference spectrum into contributions along the optically excited transition dipole in the corrin ring designated x and perpendicular to this direction, y + z . The data obtained for F 2 PhEtyCbl is compared with that for CNCbl in the S 1 minimum at time delays > 600 fs and MeCbl in the S 1 minimum at 100 ps . The ground-state XANES have been scaled to 1.25 at the high-energy peak for comparison.…”

“…Separation of the XANES difference signals into x - and y + z -contributions provides more detail. A linear combination of parallel and perpendicular spectra creates molecule-fixed X-ray spectra. − These x -polarized and y + z -polarized difference spectra allow a taxonomic approach: classifying the major contribution of the x -polarized signal as equatorial ligands and that of the y + z -polarized signal as dominated by axial ligands. The molecule-fixed difference spectra are shown in Figure and compared with the molecule-fixed difference spectra for CNCbl in the S 1 minimum (time delay > 600 fs) and MeCbl in the S 1 minimum at 100 ps . See Figure S4 for a comparison of the isotropic difference spectra and the estimated excited-state spectra.…”

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations

“…By selectively probing inner shell transitions along the direction parallel or perpendicular to the valence transition dipole, OPS-XTA introduces another level of sensitivity in electronic structure and local geometry determination to XTA. Sension and coworkers have implemented this approach to resolve the ultrafast structural relaxation of excited vitamin B 12 (35)(36)(37)(38). Previous studies by Della Longa and colleagues have partially assigned features of the MbCO and Mb XANES based on a combination of electronic structure calculations and both steady state XANES of Mb in solution (18) and through series of polarized XANES of single-crystal MbCO and cryogenically trapped Mb*CO where CO is dissociated but bound elsewhere in the heme pocket (10,39).…”