Bicarbonate binding to the non-heme iron of photosystem II investigated by Fourier transform infrared difference spectroscopy and C-13-labeled bicarbonate

The binding site of the non-heme iron of photosystem II (PS II) is investigated by light-induced Fourier tranform infrared (FTIR) difference spectroscopy on Tris-washed membranes. The nonheme iron is oxidized (Fe3+) in the dark with ferricyanide and reduced (Fe2+) after light-induced charge separation by electron transfer from the semiquinone anion Q(A)(-). EPR experiments and IR modes of ferri- and ferrocyanide show that the electron donor side of PS II is reduced in less than 2 s after a flash and that ferricyanide reoxidizes the non-heme iron with a half-time of approximate to 20 s. Recording FTIR spectra before and 2 s after flash illumination thus results in the Fe2+/Fe3+ difference spectrum. This spectrum shows band shifts and intensity changes of IR modes from ligands and neighboring residues of the nonheme iron. The IR modes of bicarbonate are revealed by comparison of Fe2+/Fe3+ spectra obtained on PS II membranes with C-12 or C-13 isotope labeled bicarbonate in H2O and in (H2O)-H-2. The nu(as)(CO) and nu(s)(CO) modes of bicarbonate in the Fe2+ state are assigned at 1530 +/- 10 and 1338 cm(-1), respectively, The low frequency of the nu(as)(CO) mode is taken as experimental evidence that bicarbonate is a ligand of the non-heme iron. Furthermore, the small frequency difference (192 cm(-1)) between the nu(as)(CO) and nu(s)(CO) modes as compared to even hydrogen-bonded ionic bicarbonate strongly indicates that bicarbonate is a bidentate ligand of the non-heme iron in PS II. Upon iron oxidation, the bicarbonate modes are largely affected. The nu(s)(CO) mode is assigned at 1228 cm(-1), while the nu(as)(CO) mode is tentatively assigned at 1658 +/- 20 cm(-1). The strong up- and downshifts of the nu(as) and nu(s) (CO) modes of bicarbonate upon iron oxidation results in a frequency difference of 430 +/- 20 cm(-1) that is not only explained by the increased charge on the iron but indicates that bicarbonate is a monodentate ligand of the oxidized iron. The sensitivity of the nu(s)(CO) mode of bicarbonate to H-1/H-2 exchange in both the Fe2+ and Fe3+ states and the presence in the Fe2+ state of a delta(COH) mode at 1258 cm(-1) confirm that bicarbonate and not carbonate is the iron ligand and further exhibits hydrogen bond(s) with the protein. The C-13 isotope-sensitive modes of bicarbonate are not affected by N-15 labeling of the PS II membranes. N-15 sensitive signals at 1111/1102 and 1094 cm(-1) are assigned to side chain modes from histidine ligands of the iron. The latter signal is proposed to account for a histidine ligand that deprotonates upon iron oxidation. The involvement of protein peptide groups and side chains in the hydrogen-bond network around the iron is also discussed.