Split Electron Paramagnetic Resonance Signal Induction in Photosystem II Suggests Two Binding Sites in the S₂ State for the Substrate Analogue Methanol

Abstract

Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so-called split signals detectable with continuous wave-electron paramagnetic resonance (CW-EPR). These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3, and S0 states [Su, J.-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or unbind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the oxygen evolving complex (OEC), thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split EPR signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site are discussed.

Interaction of methanol with the oxygen-evolving complex: atomistic models, channel identification, species dependence, and mechanistic implications

“…A hypothesis regarding the presence of two methanol binding sites was previously advanced by Sjöholm et al to account for the different methanol concentration dependence in the induction of “split” EPR signals of the OEC, that is, of states that contain a tyrosyl radical interacting with the manganese cluster. 63 The postulated interaction modes (direct binding of methanol to Mn1 and/or Mn4) can no longer be considered viable, so it might be worth revisiting that analysis in light of the present results and of the required occupation of the W6 site.…”

“…73,132,142–144 In this case, the influence of MeOH on proton release from W1 would be a second-order effect, acting through perturbation of the proton acceptor. This may result in modulation of the relative stabilities of intermediates such as tyrosyl radical states 53,57,58,63,122,145,146 in a species-dependent manner and is a subject that deserves further study.…”

“…62 The concept of direct ligation to the Mn1 and Mn4 sites with different affinities was recently used also in the interpretation of D1-Tyr161 radical “split signals” that can be generated from the S 2 state under different illumination conditions. 63 On the other hand, the ESEEM study of the S 0 state did not result in observation of specific interactions, disfavoring direct methanol binding to the OEC. 78 It is clear that neither the data can be reconciled, nor any of the interpretations supported with confidence.…”

“…78 It is clear that neither the data can be reconciled, nor any of the interpretations supported with confidence. Beyond the intrinsic limitations in the treatment of deuteron dipolar couplings, the involvement of Mn1 as a potential binding site 49,59,63 conflicts with channel accessibility studies, 20,25 while explanations that invoke drastic reorganizations of first-sphere ligands seem incompatible with the small observed spectroscopic perturbations and the retention of catalytic activity. Similar ambiguities pertain to structural interpretations of methanol effects on the miss parameter for S-state advancement.…”

Pulse Electron Paramagnetic Resonance Studies of the Interaction of Methanol with the S2State of the Mn4O5Ca Cluster of Photosystem II

“…35 There appear to be differences in the concentration dependence of the effects of methanol on these signals that are S-state dependent, indicating that the binding affinity of MeOH at the Mn 4 CaO 5 cluster may change as a function of the oxidation state of the cluster. 34 …”