Iron Bound to the High-Affinity Mn-Binding Site of the Oxygen-Evolving Complex Shifts the pK of a Component Controlling Electron Transport via Y_Z

Abstract

Flash-probe fluorescence spectroscopy was used to compare the pH dependence of charge recombination between Y(Z)(*) and Q(a)(-) in Mn-depleted, photosystem II membranes [PSII(-Mn)] and in membranes with the high-affinity (HA(Z)) Mn-binding site blocked by iron [PSII(-Mn,+Fe); Semin, B. K., Ghirardi, M. L., and Seibert, M. (2002) Biochemistry 41, 5854-5864]. The apparent half-time for fluorescence decay (t(1/2)) in PSII(-Mn) increased from 9 ms at pH 4.4 to 75 ms at pH 9.0 [with an apparent pK (pK(app)) of 7.1]. The actual fluorescence decay kinetics can be fit to one exponential component at pH <6.0 (t(1/2) = 9.5 ms), but it requires an additional component at pH >6.0 (t(1/2) = 385 ms). Similar measurements with PSII(-Mn,+Fe) membranes show that iron binding has little effect on the maximum and minimum t(1/2) values measured at alkaline and acidic pHs but that it does shift the pK(app) from 7.1 to 6.1 toward the more acidic pK(app) value typical of intact membranes. Light-induced Fe(II) blocking of the PSII(-Mn) membrane is accompanied by a decrease in buffer Fe(II) concentration. This decrease was not the result of Fe(II) binding, but rather of its oxidation at two sites, the HA(Z) site and the low-affinity site. Mössbauer spectroscopy at 80 K on PSII(-Mn,+Fe) samples, prepared under conditions providing the maximal blocking effect but minimizing the amount of nonspecifically bound iron cations, supports this conclusion since this method detected only Fe(III) cations bound to the membranes. Correlation of the kinetics of Fe(II) oxidation with the blocking parameters showed that blocking occurs after four to five Fe(II) cations were oxidized at the HA(Z) site. In summary, the blocking of the HA(Z) Mn-binding site by iron in PSII(-Mn) membranes not only prevents the access of exogenous donors to Y(Z) but also partially restores the properties of the hydrogen bond net found in intact PS(II), which in turn controls the rate of electron transport to Y(Z).

Iron-Blocking the High-Affinity Mn-Binding Site in Photosystem II Facilitates Identification of the Type of Hydrogen Bond Participating in Proton-Coupled Electron Transport via YZ•

“…Determinations of activation energies in PSII(-Mn) membranes were done in buffer A (pH 5.5 and 6.5) and in Tricine buffer containing 50 mM Tricine/NaOH (pH 8.2), 15 mM NaCl, and 0.4 M sucrose. No differences in the decay kinetics of the samples in either MES or Tricine were observed if the buffers were poised at the same pH (pH 7.0) (23). Iron-blocked PSII(-Mn) membranes [note that Fe-blocking of PSII(-Mn) membranes was performed in buffer A at pH 6.5 prior to examination at other pHs] were examined using MES buffer (50 mM MES/ NaOH, 15 mM NaCl, and 0.4 M sucrose) at pHs of 5.5, 6.5, and 8.2.…”

“…Fluorescence decay kinetics approximated by a single (fast) exponential component were calculated using the equation, y ) y 0 + a f e -k f t . In the case of PSII(-Mn) or PSII(-Mn, +Fe), the samples were measured at pH 5.5, but PSII(-Mn) samples were also measured at pH 6.5 (23). Fluorescence decay kinetics of PSII(-Mn, +Fe) samples measured at pH 6.5 or PSII(-Mn) samples measured at pH 8.2 were approximated using the equation, y ) y 0 + a f e -k f t + a s e -k s t , because two decay components (fast and slow) provided a better fit to the data (23).…”

“…It is widely accepted that deprotonation of Y Z during oxidation occurs via a hydrogen bond between Y Z and a nearby group X (14) [or base B (15); for reviews see refs 1, 5, 16, 17]. Spectroscopic studies (12,18) and numerous kinetic investigations of the rate of electron transfer from Y Z to P680 + in the forward direction (10,15,19,20) or from P680 to Y Z • in the back reaction (21)(22)(23) reveal that Y Z • is hydrogenbonded in PSII(-Mn) preparations. Recent studies indicate the involvement of only a single proton during the ratelimiting step of the Y Z oxidation (24) or Y Z • Q a -recombination (23) processes.…”

“…The mutually exclusive interaction of Fe(II) and Mn(II) cations with the HA Z site was originally recognized using the DPC-inhibition assay in a series of kinetic studies (38). However, we recently found that incubation of Mn-depleted PSII membranes with Fe(II) under weak light [PSII(-Mn, +Fe) preparations] for a short period of time (in a process similar to photoactivation) leads to the light-dependent oxidation of Fe(II) and the formation of an iron cluster with 2 e n e 4.5 (23). Thus, after incubation, the interaction of one iron cation with the donor side of PSII(-Mn) membranes is accompanied by the creation of a new Fe-binding site (37).…”

“…Thus, after incubation, the interaction of one iron cation with the donor side of PSII(-Mn) membranes is accompanied by the creation of a new Fe-binding site (37). The resultant purported iron cluster that is formed blocks the donation of electrons to Y Z • from a number of exogenous electron donors [i.e., DPC, Mn(II), Fe(II)] (37) and shifts the pK app of the hydrogen bond partner for Y Z to more acidic pH (23). Modification of these properties (the accessibility of Y Z • for exogenous donors and the shift of the pK app ) by the coordination of Fe(III) indicates that properties assigned to the donor side of intact PSII material are partially reconstituted by iron cations bound to PSII(-Mn) membranes.…”