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.

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.