Time-Resolved Spectroscopic Studies of B₁₂ Coenzymes:  The Photolysis and Geminate Recombination of Adenosylcobalamin

Abstract

Femtosecond transient absorption spectroscopy has been used to investigate the photolysis of coenzyme B12 (5‘-deoxyadenosylcobalamin). Transient kinetic measurements obtained at wavelengths between 400 and 633 nm were analyzed globally to obtain time constants. Transient spectral data obtained for time delays between 5 ps and 9 ns were analyzed by matrix decomposition to identify distinct spectral components present in the data. Photoexcitation results in homolysis of the carbon−cobalt bond forming a singlet radical pair on a picosecond time scale. The subsequent spectral changes probe conformational relaxation and geminate recombination. Analysis of the spectral data suggests that 76 ± 4% of the geminate radical pairs recombine, resulting in a quantum yield of 0.24 ± 0.04 for the formation of solvent separated radicals, in good agreement with literature values of 0.20 ± 0.03 and 0.23 ± 0.04 [Chen, E.; Chance, M. R. Biochemistry 1993, 32, 1480−1487]. The geminate recombination of the adenosyl radical with cob(II)alamin occurs biphasically with exponential time constants of 150 ± 20 ps and 0.5 ± 0.2 ns. The effective recombination rate from a single-exponential fit to the data is (0.250 ns)-1 = 4 ns-1.

Protein Motions Are Coupled to the Reaction Chemistry in Coenzyme B12‐Dependent Ethanolamine Ammonia Lyase

“…[20] Kinetic analysis of these data fit to the sum of four exponentials, in close agreement with similar UV/Vis kinetic measurements. [28] Selected TRIR difference spectra acquired between 1 ps-2 ns from the EAL holoenzyme ( in the EAL measurements, both of which coincide with the protein amide I band and decay with similar kinetics to the signal from the cofactor. There are also differences around 1425-1475 cm À1 , which coincide with the amide II bands of deuterated proteins.…”

Excited electronic states and internal conversion in cyanocobalamin

“…Ultrafast broadband transient absorption spectroscopy has been used to study the photochemistry and photophysics of naturally occurring and synthetically prepared cobalamin compounds [9,[15][16][17][18][19][20][21][22][23][24][25][26][27]. Alkylcobalamins are photolabile.…”

“…Alkylcobalamins are photolabile. For most alkylcobalamins that have been studied to date, the primary photolysis quantum yield is on the order of unity [15,21,22,24]. The quantum yield for long-lived radicals is determined by competition between diffusive separation of the radical pair and geminate recombination [15].…”

Time-Resolved Spectroscopic Studies of B12 Coenzymes: A Comparison of the Primary Photolysis Mechanism in Methyl-, Ethyl-, n-Propyl-, and 5‘-Deoxyadenosylcobalamin

“…[24][25][26] The principal photoproduct observed following excitation of adenosylcobalamin at 400 or 520 nm is cob(II)alamin. Unlike methylcobalamin, the photolysis quantum yield in adenosylcobalamin is determined .…”