Ultrafast X-ray Absorption Spectroscopy Reveals Excited-State Dynamics of B12 Coenzymes Controlled by the Axial Base.

Polarized time-resolved X-ray absorption spectroscopy at the Co K-edge is used to probe the excited-state dynamics and photolysis of base-off methylcobalamin and the excited-state structure of base-off adenosylcobalamin. For both molecules, the final excited-state minimum shows evidence for an expansion of the cavity around the Co ion by ca. 0.04 to 0.05 Å. The 5-coordinate base-off cob(II)alamin that is formed following photodissociation has a structure similar to that of the 5-coordinate base-on cob(II)alamin, with a ring expansion of 0.03 to 0.04 Å and a contraction of the lower axial bond length relative to that in the 6-coordinate ground state. These data provide insights into the role of the lower axial ligand in modulating the reactivity of B12 coenzymes.

Watching Excited State Dynamics with Optical and X-ray Probes: The Excited State Dynamics of Aquocobalamin and Hydroxocobalamin.

Femtosecond time-resolved X-ray absorption (XANES) at the Co K-edge, X-ray emission (XES) in the Co Kß and valence-to-core regions, and broadband UV-vis transient absorption are combined to probe the femtosecond to picosecond sequential atomic and electronic dynamics following photoexcitation of two vitamin B12 compounds, hydroxocobalamin and aquocobalamin. Polarized XANES difference spectra allow identification of sequential structural evolution involving first the equatorial and then the axial ligands, with the latter showing rapid coherent bond elongation to the outer turning point of the excited state potential followed by recoil to a relaxed excited state structure. Time-resolved XES, especially in the valence-to-core region, along with polarized optical transient absorption suggests that the recoil results in the formation of a metal-centered excited state with a lifetime of 2-5 ps. This combination of methods provides a uniquely powerful tool to probe the electronic and structural dynamics of photoactive transition-metal complexes and will be applicable to a wide variety of systems.

Transmission Mode 2D-IR Spectroelectrochemistry of In Situ Electrocatalytic Intermediates.

Unraveling electrocatalytic mechanisms, as well as fundamental structural dynamics of intermediates, requires spectroscopy with high time and frequency resolution that can account for nonequilibrium in situ concentration changes inherent to electrochemistry. Two-dimensional infrared (2D-IR) spectroscopy is an ideal candidate, but several technical challenges have hindered development of this powerful tool for spectroelectrochemistry (SEC). We demonstrate a transmission-mode, optically transparent thin-layer electrochemical (OTTLE) cell adapted to 2D-IR-SEC to monitor the important Re(bpy)(CO)3Cl CO2-reduction electrocatalyst. 2D-IR-SEC reveals pronounced differences in both spectral diffusion time scales and spectral inhomogeneity in the singly reduced catalyst, [Re(bpy)(CO)3Cl]•-, relative to the starting Re(bpy)(CO)3Cl. Cross-peaks between well-resolved symmetric vibrations and congested low-frequency bands enable direct assignment of all distinct species during the electrochemical reaction. With this information, 2D-IR-SEC provides new mechanistic insights regarding unproductive, catalyst-degrading dimerization. 2D-IR-SEC opens new experimental windows into the electrocatalysis foundation of future energy conversion and greenhouse gas reduction.

The Photoactive Excited State of the B12-Based Photoreceptor CarH.

We have used transient absorption spectroscopy in the UV-visible and X-ray regions to characterize the excited state of CarH, a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light. With visible excitation, a nanosecond-lifetime photoactive excited state is formed with unit quantum yield. The time-resolved X-ray absorption near edge structure difference spectrum of this state demonstrates that the excited state of AdoCbl in CarH undergoes only modest structural expansion around the central cobalt, a behavior similar to that observed for methylcobalamin rather than for AdoCbl free in solution. We propose a new mechanism for CarH photoreactivity involving formation of a triplet excited state. This allows the sensor to operate with high quantum efficiency and without formation of potentially dangerous side products. By stabilizing the excited electronic state, CarH controls reactivity of AdoCbl and enables slow reactions that yield nonreactive products and bypass bond homolysis and reactive radical species formation.

Ultrafast XANES Monitors Femtosecond Sequential Structural Evolution in Photoexcited Coenzyme B12.

Polarized X-ray absorption near-edge structure (XANES) at the Co K-edge and broadband UV-vis transient absorption are used to monitor the sequential evolution of the excited-state structure of coenzyme B12 (adenosylcobalamin) over the first picosecond following excitation. The initial state is characterized by sub-100 fs sequential changes around the central cobalt. These are polarized first in the y-direction orthogonal to the transition dipole and 50 fs later in the x-direction along the transition dipole. Expansion of the axial bonds follows on a ca. 200 fs time scale as the molecule moves out of the Franck-Condon active region of the potential energy surface. On the same 200 fs time scale there are electronic changes that result in the loss of stimulated emission and the appearance of a strong absorption at 340 nm. These measurements provide a cobalt-centered movie of the excited molecule as it evolves to the local excited-state minimum.

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

Polarized transient X-ray absorption near-edge structure (XANES) was used to probe the excited-state structure of a photostable B12 antivitamin (Coß-2-(2,4-difluorophenyl)-ethynylcobalamin, F2PhEtyCbl). A drop-on-demand delivery system synchronized to the LCLS X-ray free electron laser pulses was implemented and used to measure the XANES difference spectrum 12 ps following excitation, exposing only ∼45 µL of sample. Unlike cyanocobalamin (CNCbl), where the Co-C bond expands 15-20%, the excited state of F2PhEtyCbl is characterized by little change in the Co-C bond, suggesting that the acetylide linkage raises the barrier for expansion of the Co-C bond. In contrast, the lower axial Co-NDMB bond is elongated in the excited state of F2PhEtyCbl by ca. 10% or more, comparable to the 10% elongation observed for Co-NDMB in CNCbl.

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

We use picosecond time-resolved polarized X-ray 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 B12). 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 [ 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.

Ultrafast X-ray Absorption Near Edge Structure Reveals Ballistic Excited State Structural Dynamics.

Polarized ultrafast time-resolved X-ray absorption near edge structure (XANES) allows characterization of excited state dynamics following excitation. Excitation of vitamin B12, cyanocobalamin (CNCbl), in the αß-band at 550 nm and the γ-band at 365 nm was used to uniquely resolve axial and equatorial contributions to the excited state dynamics. The structural evolution of the excited molecule is best described by a coherent ballistic trajectory on the excited state potential energy surface. Prompt expansion of the Co cavity by ca. 0.03 Å is followed by significant elongation of the axial bonds (>0.25 Å) over the first 190 fs. Subsequent contraction of the Co cavity in both axial and equatorial directions results in the relaxed S1 excited state structure within 500 fs of excitation.