Time-Resolved Spectroscopic Studies of B₁₂Coenzymes:  The Identification of a Metastable Cob(III)alamin Photoproduct in the Photolysis of Methylcobalamin

Abstract

Ultrafast transient absorption spectroscopy has been used to investigate the primary photochemistry of methylcobalamin. Approximately 27% of the initially excited methylcobalamin undergoes a bond homolysis on a subpicosecond time scale. The remaining 73% forms a metastable photoproduct with a spectrum similar to that of cob(III)alamin compounds. The ultraviolet absorption spectrum of the metastable photoproduct exhibits a prominent γ-band at 340 nm, characteristic of a cob(III)alamin with a very weak axial ligand. The metastable photoproduct recovers to the ground electronic state of methylcobalamin on a 1.2 ± 0.5 ns time scale, leaving only cob(II)alamin (and presumably methyl radical) at 9 ns. The primary photochemical yield of cob(II)alamin is determined largely by the branching ratio between the two photoproduct channels. A 40 ps transient absorption difference spectrum of methylcobalamin bound to methionine synthase indicates that the branching ratio and initial production of cob(II)alamin is not changed in the enzyme-bound cofactor. The substantial photolysis protection afforded by the enzyme must be attributed to structural and electronic effects which enhance the intrinsic rate of recombination of the radical pair, rather than to suppression of primary bond homolysis.

New light on vitamin B12: The adenosylcobalamin-dependent photoreceptor protein CarH

“…For example, it is stated 56 that the spectrum of Intermediate C appears to be similar to the S 1 state seen in the photolysis of free MeCbl 601,61 . The S 1 state in MeCbl photolysis has now been characterised as a d/p → p* metal-to-ligand charge-transfer state 61 (consistent with theoretical density functional theory calculations) 62 , but was originally considered as 'a cob(III)alamin with a very weak axial ligand' 60 . The corresponding theoretical calculations for AdoCbl have not been completed to date but steady progress is being made with such calculations.…”

Theoretical investigations of photophysics and spectroscopic properties in B12 cofactors.

“…56,58,60,65 To The dynamics of the photoinduced homolysis of B 12 cofactors were investigated on a time scale ranging from femtoseconds to nanoseconds, allowing for the detailed mechanism of Co-C bond photolysis to be obtained. In the case of CNCbl, it was found that the metastable S 1 state has a ligand-to-metal charge transfer (LMCT)…”

“…However, the excitation at 520 nm was followed only by formation of a long-lived S 1 state. 56,58,65 In contrast, the photolysis of AdoCbl was found to be wavelength independent resulting in a creation of a geminate radical pair within 100-130 ps at room temperature. 57,59,62 The same studies for ethyland n-propylcobalamin indicated the formation of radical pair when samples were excited at both 400 and 520 nm but the rapid dissociation contribution was mainly observed following the higher energy excitation.…”

“…The most recent results originate from the TAS studies by Sension et al 56,58,60,65 and the spectroscopic studies by…”

“…Whereas the metastable S 1 state in CNCbl was assigned as corresponding to ligand-to-metal charge transfer (LMCT) transition, 64 the S 1 in MeCbl was described as metal-to-ligand charge transfer (MLCT). 56,58,60 On the other hand, the S 1 for coenzyme B 12 presented different character (MLCT or LMCT) depending on the environment. 65 In the light of the above findings, the photochemistry of cobalamins was further explored with the aim of theoretical studies.…”