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 non-heme iron is oxidized (Fe3+) in the dark with ferricyanide and reduced (Fe2+) after light-induced charge separation by electron transfer from the semiquinone anion QA-. 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 approximately 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 non-heme iron. The IR modes of bicarbonate are revealed by comparison of Fe2+/Fe3+ spectra obtained on PS II membranes with 12C or 13C isotope labeled bicarbonate in H2O and in 2H2O. 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 1H/2H 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 13C isotope-sensitive modes of bicarbonate are not affected by 15N labeling of the PS II membranes. 15N 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.

In order to investigate the changes of protonation or environment of carboxylic residues occurring upon photoreduction of the secondary quinone acceptor (QB) in the reaction center (RC) of the photosynthetic bacteria Rhodobacter sphaeroides 2.4.1., we have performed light-induced Fourier transform infrared (FTIR) spectroscopy on RCs from wild-type (Wt) and several site-directed mutants. The FTIR QB-/QB spectra have been obtained at pH 7 upon single-saturating flash excitation for native RCs and RC mutants containing either a single-site mutation, with Gln at L212 (EQ L212), Asn at L213 (DN L213), or Asn at L210 (DN L210), or a double-site mutation with both Gln at L212 and Asn at L213 (EQ L212 + DN L213). The assignment of an IR band to the protonation/deprotonation of a particular carboxylic side chain was analyzed by combining the effects of site-directed mutagenesis and 1H/2H isotope exchange. A positive band at 1728 cm-1 in the QB-/QB spectra was observed in Wt, DN L213, and DN L210 and was absent in the mutants EQ L212 and EQ L212 + DN L213. The intensity of the 1728 cm-1 band was significantly reduced in 2H2O, and a new feature appears at 1717 +/- 1 cm-1. Furthermore, the amplitude of the 1728 cm-1 band was similar in native and DN L210 RCs but was increased in DN L213. This band is attributed to partial proton uptake by Glu L212 estimated to be 0.3-0.4 H+/QB- in native and DN L210 RCs and O.5-0.6 H+/QB- in DN L213 RCs. In contrast, the FTIR QB-/QB spectra show no evidence for change of protonation or environment of Asp L213 upon QB- formation. The increased protonation of Glu L212 in DN L213 RCs is explained by a decreased Glu L212 pKa value due to the loss of a negatively charged Asp L213. Part of a small differential signal at 1732 (+)/1740 (-) cm-1 that is affected by 1H/2H exchange is tentatively assigned to an environmental shift of the protonated Asp L210. A negative signal at 1685 cm-1 is propose to arise from the absorption change of the amide I carbonyl mode of Glu L212.(ABSTRACT TRUNCATED AT 400 WORDS)

Fourier transform infrared (FTIR) spectroscopy probes the vibrational properties of amino acids and cofactors, which are sensitive to minute structural changes. The lack of specificity of this technique, on the one hand, permits us to probe directly the vibrational properties of almost all the cofactors, amino acid side chains, and of water molecules. On the other hand, we can use reaction-induced FTIR difference spectroscopy to select vibrations corresponding to single chemical groups involved in a specific reaction. Various strategies are used to identify the IR signatures of each residue of interest in the resulting reaction-induced FTIR difference spectra. (Specific) Isotope labeling, site-directed mutagenesis, hydrogen/deuterium exchange are often used to identify the chemical groups. Studies on model compounds and the increasing use of theoretical chemistry for normal modes calculations allow us to interpret the IR frequencies in terms of specific structural characteristics of the chemical group or molecule of interest. This review presents basics of FTIR spectroscopy technique and provides specific important structural and functional information obtained from the analysis of the data from the photosystems, using this method.