Structural Evolution of Photoexcited Methylcobalamin toward a CarH-like Metastable State: Evidence from Time-Resolved X-ray Absorption and X-ray Emission.

CarH is a protein photoreceptor that uses a form of B, adenosylcobalamin (AdoCbl), to sense light via formation of a metastable excited state. Aside from AdoCbl bound to CarH, methylcobalamin (MeCbl) is the only other example─to date─of photoexcited cobalamins forming metastable excited states with lifetimes of nanoseconds or longer. The UV-visible spectra of the excited states of MeCbl and AdoCbl bound to CarH are similar.

The Photoactive Excited State of the B-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 B, 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.

Antivitamins B 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 B antivitamin (Coβ-2-(2,4-difluorophenyl)-ethynylcobalamin, FPhEtyCbl). 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 FPhEtyCbl 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.

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 B). 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.