Interpretation of fluorescence kinetics

The kinetics of QA- oxidation as measured by fluorescence show different rates after the first and second flashes, reflecting the two different reactions of the two-electron gate.

QA.QB -> QA-QB = QAQB-          (1)

QAQB- -> QA-QB- = QAQBH2       (2)

The rate after the first flash is a convolution of two processes. The fast phase (150- 200µs) is due to centers in which QB is bound before the flash. The slow phase (1 - 2ms) reflects centers in which QB is not bound before the flash so that electron transfer has to wait for Q binding.

The equilibrium and rate constants for the forward and reverse electron transfer and for binding and dissociation of plastoquinone at the QB site can be calculated using the following assumptions:

  1. The ratio of amplitudes of fast and slow components of the rapid biphasic decay kinetics of QA- following an actinic flash was assumed to reflect the ratio of the initial fraction of QA.QB to QA.[vacant] centers.
  2. The dissociation constant of plastoquinone from the QB site in the dark was assumed to remain unchanged after an actinic flash.
  3. Binding of plastoquinone to the QB-site was assumed to follow a pseudo-first order reaction kinetics when the pool was initially oxidized (ten-fold excess of oxidized plastoquinone over reaction center).

The two following equations are solutions of the rate equations derived from the model.

r1 + r2 = kon ( 1 + Ao / Bo ) + kBA{ 1 + Kapp ( 1 + Ao / Bo ) }          (1)

r1 . r2 = kon . kBA ( 1 + Ao / Bo ) ( 1 + Kapp )          (2)

Variables r1 and r2 are the slow and fast rate constants obtained directly from the experimental data by fitting the kinetic traces of fluorescence decay after one actinic flash to two exponential components, and a residual; Ao and Bo are the amplitudes of the slow and fast component, respectively, of the fitted kinetic traces, also obtained from the two-exponential fit. KO, the plastoquinone dissociation constant, is calculated as the ratio Ao/Bo (assumption i) above).

Kapp is the apparent equilibrium constant for sharing an electron between QA and QB, defined (if protonated states are included) as:

                      [QAQB-] + [QAQB-(H+)]
Kapp = -------------------------------------------------
            [QA-] + [QA-(H+)] + [QA-QB] + [QA-QB(H+)]

For simplicity, we can ignore the effects of protonation, and treat Kapp at any fixed pH as determined by the equilibrium constants for electron transfer, and the dissociation of Q from the site:

Kapp = -------------------
             [QA-] + [QA-QB]

         = KE / (1 + KO)          (3)

Again, for simplicity we have ignored the contribution of the quinone pool, so that

KO = QA- / QA-.QB

      = Ao / Bo

      = koff / kon


KE = QA.QB- / QA-.QB

      = kAB / kBA

so that

Kapp = kAB / kBA . ( 1 + Ao / Bo ) -1          (3')

The apparent equilibrium constant, Kapp, can be determined experimentally from the ratio of the half-time of the back reaction S2PQAQB- -> S1PQAQB to that of the back reaction S2PQA- -> S1PQA. The measured parameters therefore allow us to determine the rate constants and equilibrium constants which define the two-electron gate mechanism.

By repeating the above experiments at different pH values, we can also determine the pK values which describe the role of protons in the mechanism, but this more extensive data set has so far been collected only in A. hybridis and its S264G mutant (7).

©Copyright 1996, Antony Crofts, University of Illinois at Urbana-Champaign,