Abstract
The quantum yield and kinetics of decay of cob(II)alamin formed by pulsed-laser photolysis of adenosylcobalamin (AdoCbl; coenzyme B(12)) in AdoCbl-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied on the 10(-7)-10(-1) s time scale at 295 K by using transient ultraviolet-visible absorption spectroscopy. The aim is to probe the mechanism of formation and stabilization of the cob(II)alamin-5'-deoxyadenosyl radical pair, which is a key intermediate in EAL catalysis, and the influence of substrate binding on this process. Substrate binding is required for cobalt-carbon bond cleavage in the native system. Photolysis of AdoCbl in EAL leads to a quantum yield at 10(-7) s for cob(II)alamin of 0.08 +/- 0.01, which is 3-fold smaller than for AdoCbl in aqueous solution (0.23 +/- 0.01). The protein binding site therefore suppresses photoproduct radical pair formation. Three photoproduct states, P(f), P(s), and P(c), are identified in holo-EAL by the different cob(II)alamin decay kinetics (subscripts denote fast, slow, and constant, respectively). These states have the following first-order decay rate constants and quantum yields: 2.2 x 10(3) s(-1) and 0.02 for P(f), 4.2 x 10(2) s(-1) and 0.01 for P(s), and constant amplitude, with no recombination, and 0.05 for P(c), respectively. Binding of the substrate analogue (S)-1-amino-2-propanol to EAL eliminates the P(f) state and lowers the quantum yield of P(c) (0.03) relative to that of P(s) (0.01) but does not significantly change the quantum yield or decay rate constant of P(s), relative to those of holo-EAL. The substrate analogue thus influences the quantum yield at 10(-7) s by changing the cage escape rate from the geminate cob(II)alamin-5'-deoxyadenosyl radical pair state. However, the predicted substrate analogue binding-induced increase in the quantum yield is not observed. It is proposed that the substrate analogue does not induce the radical pair stabilizing changes in the protein that are characteristic of true substrates.