THE TWO-ELECTRON GATE OF REACTION CENTERS
Following illumination of bacterial reaction centers in the open state,
the following sequence of reactions occurs:
P.QA.QB --> P*.QA.QB
--> P+.QA-.QB <=>
kAP = 10 s-1
The photochemical products return to the ground state by back-reaction through a pathway which has a half-time (in reaction centers from Rb. sphaeroides) of about 70 -100 ms, when forward electron transfer from QA- is prevented. When forward electron transfer can occur to the secondary quinone, the back- reaction time is longer, because the electron in the acceptor pool is shared between QA and QB. The ratio of back-reaction times is related to the fractional occupancy of the QA-.QB state.
Relation between back-reaction rate constants and the equilibrium constant
between the acceptor quinones can be approached as follows.
We start with the simplest case. Consider the reaction between QA
and QB when QB is bound.
QA-.QB <=> QA.QB-
with equilibrium constant:
KE = ------------
We normalize by setting the total reaction center concentration to 1
in arbitrary units. Then the concentration of QA-
formed on a saturating flash when forward electron transfer is prevented
will be 1. We can prevent forward electron transfer by adding an inhibitor
which binds instead of QB, or by preparing reaction centers
without QB. Under these circumstances, the back- reaction, measured
by disappearance of P+, will occur according to the following
rate equation (P+ has been omitted from the right-hand term
vA = -d[P+]/dt = kAP.[QA-]
for the initial rate,
vAinit = kAP.[QA-]tot
Apparent rate constant will be the same as the true rate constant, kAP
In the presence of QB, the probability of an electron being
on QA will be less because of the electron transfer reaction
above, and will be described by the equilibrium constant KE.
Since equilibrium is rapid compared to the back-reaction, we can use the
relative rates of the back-reactions under the two conditions to measure
Let f be the fraction of centers in which the electron is on QA.
Assuming rapid equilibration, immediately after a saturating flash we will
f = [QA-.QB]/[reaction center]tot
KE = (1 - f) / f
f = 1/(1 + KE)
The rate of the back-reaction will be given by
vB = -d[P+]/dt = kAP.[ QA-.QB]
for initial rate,
vBinit = kAP.f = kAP / (1
The apparent rate constant will be changed to kAP / (1 +
KE). Since the reaction is first-order, the half-times under
both conditions will depend on the apparent rate constants, so that
vAinit / vBinit = t½(B)
/ t½(A) = 1 + KE
KE = t½(B) / t½(A) -
The ratio of the back-reaction half-times (or apparent rate constants)
therefore provides a direct measurement of the equilibrium constant for
the sharing of an electron between primary and secondary acceptors.
The true situation is more complicated, because of the need to take
account of the following secondary reactions:
i) The fraction of centers with QB bound before the flash
will be less than 1 because of the reaction
QA.[vacant] + Qpool <=> QA.QB
ii) Formation of QB- is stabilized by binding
of a proton to a neighboring group on the protein, according to the reaction
QA.QB- + H+ <=> QA.QB-
iii) Reactions on the donor side may consume P+. A treatment
similar to that for the acceptor side discussed above can be used to probe
equilibria between primary and secondary donors.
Nevertheless, the above treatment can be readily extended to the more
complicated situation, and has provided much information about equilibrium
constants among components in acceptor and donor pools.