Early photocycle kinetic behavior of the E46A and Y42F mutants of photoactive yellow protein: Femtosecond spectroscopy

In the photoactive yellow protein, PYP, both Glu46 and Tyr42 form hydrogen bonds to the phenolic OH group of the p-hydroxycinnamoyl chromophore. Previous work on replacement of the carboxyl group of Glu46 by an amide group (Glu46Gln) has shown that changing the nature of this hydrogen bond has a minimal effect on the rate constant for the formation of the first intermediate (I_o) and on the excited state lifetime, whereas the rate constants for the formation of the second (I_o^≠) and third (I₁) intermediates were increased by factors of ∼30 and 5, respectively. In the present experiments, two additional mutants (Glu46Ala and Tyr42Phe) have been studied. These two mutants are shown to behave kinetically very similarly to one another. In both cases, the rate constant for I_o formation is decreased by a factor of ∼2, with little or no effect on the photochemical yield as a consequence of a compensating increase in the excited state lifetime. Although we are unable to resolve the rate constant for the formation of the second intermediate from that of the first intermediate, the rate constant for the formation of the third intermediate is increased by a factor of ∼100. The structural implications of these results are discussed.