STRUCTURE AND MECHANISM OF PHOTOSYNTHETIC WATER OXIDATION
In higher plants and cyanobacteria, photosystem II (PSII) reaction centers couple light-induce charge separation with the oxidation of water to molecular oxygen and reduction of the plastoquinone acceptors, QA and QB. The water oxidation chemistry occurs in an oxygen-evolving complex (OEC), consisting of a (Mn)4-Ca cluster and a redoxactive tyrosine residue, TyrZ. Several structural models for photosynthetic water oxidation have been proposed in the literature. Our ability to test these various models, however, is limited by a lack of appropriately incisive experimental tool. During my postdoctoral work in the late Professor Jerry Babcock's lab at Michigan State University, my colleagues and I have developed low-frequency Fourier transform infrared (FTIR) difference techniques so that metal-ligand vibration can be
detected in systems, like (Mn)4-Ca site, in PSII. This work was also the first report of a low-frequency, metal-ligand vibrational mode in proteins that has been identified by FTIR. Currently, we are combining FTIR and EPR (Electron Paramagnetic Resonance) spectroscopic techniques with site-directed mutagenesis and isotopic labeling methods as an integrated approach to study structure and mechanism of photosynthetic water oxidation. With the help of recent high-resolution X-ray crystal-structures of PSII (up to 3.2 Å resolution), our approach is able to address structural and mechanistic questions of photosynthetic water oxidation at the molecular level.
The structural model of the oxygen-evolving complex in PSII [Rutherford and bousaac (2004) Science 303, 1782-1784].
Locations of electron transfer cofactors in PSII [Ferreira et al(2004) Science 303, 1831-1838].
PROGRESS AND ACCOMPLISHMENTS
(1) Substrate-binding properties of the OEC in PSII. NH3 is a structural analog of substrate H2O and an inhibitor to the water oxidation reaction in PSII, making it a valuable probe for structural properties of the substratebinding site on the OEC. We accomplished the first S2QA-/S1QA FTIR difference spectrum of NH3 treated PSII samples. We found that NH3 induced the up-shift of the 1365 cm-1 carboxylate mode in the S2QA-/S1QA FTIR difference spectra. Our results suggest that this NH3-induced
FTIR spectral change of the OEC is caused by the binding of NH3 to the Mn site on the OEC that gives rise to the altered S2 state multiline EPR signal. In addition, our results suggest that ethylene glycol acts directly or indirectly to decrease the affinity or limit the accessibility of NH3 and CH3NH2 to the Mn site on the OEC. We also found that methanol is unable to compete with NH3 on binding to the Mn site of the OEC. We are currently continuing this FTIR approach to study substrate-binding properties of the OEC in the higher S states.
(2) Structure and function of cytochrome b559 in PSII. Cytochrome (cyt) b559 was proposed to play an important role on the protection of PSII under photoinhibition conditions. However, the structural and functional studies of cyt b559 have been hindered by lack of approporiate cyt b559 mutants that still accumulated stable PSII. An early site-directed mutagenesis work by using cyanobacterium Synechocystis 6803 G showed that His22Leu mutations on the axial ligands (His22) of the heme of the £\ or £] subunits severely affected the assembly or stability of PSII complex. In addition, the other site-directed mutagenesis work by using the green alga Chlamydomonas showed that His22Tyr and His22Met mutants of the cyt b559 £\ subunit were only able to accumulate 10-15% PSII content compared to wild-type cells; however, the heme-binding pockets in these two mutants were disrupted. To overcome this problem, through collaborations with Professor Richard Debus in the University of California at Riverside, we have constructed a series of site-directed mutants on the heme ligands (His-22 of £\ and £] subunits) of cyt b559 by using Synechocystis 6803 G. We have successfully identified a couple of mutants that can grow photoautotrophically and contain the stable PSII. Further structural and functional analysis of these cyt b559 mutants and their PSII particles are in the progress.
Ammonia induces structural changes of the OECas revealed by the light-induced FTIR difference spectroscopy.
Visit of Professor Gary Brudvig couples at our institute (on 10/13/2005)
Chu HA, Sackett H and Babcock GT. 2000. Identification of a Mn-O-Mn cluster vibrational mode of the oxygen-evolving complex in photosystem II by low-frequency FTIR spectroscopy. Biochemistry 39, 14371-14376.
Chu HA, Gardner MT, Hillier W and Babcock GT. 2000. Low-frequency Fourier transform infrared spectroscopy of the oxygen-evolving complexes in photosystem II. Photosyn Res 66, 57-63.
Chu HA, Hillier W, Law NA, Sackett H, Haymond S and Babcock GT. 2000. Light-induced FTIR spectroscopy of the S2-to-S3 state transition of the oxygen-evolving complex in photosystem II. Biochim. Biophys. Acta 1459, 528-532.
Chu HA, Hillier W, Law NA and Babcock GT. 2001. Vibrational spectroscopy of the oxygen-evolving complex and of manganese model compounds. Biochim. Biophys. Acta 1503: 69-82. [Review article]
Chu HA, Debus RJ and Babcock GT. 2001. D1-Asp 170 is structurally coupled to the oxygen-evolving complex in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy. Biochemistry 40, 2312-2316.
Chu HA, Hillier W. and Debus R.J. 2004. Evidence that the C-Terminus of the D1 polypeptide of photosystem II is ligated to the manganese ion that undergoes oxidation during the S1 to S2 transition: An isotope-editing FTIR study. Biochemistry 43, 3152-3166.
Chu HA*, Feng YW, Wang CM, Chiang KA. and Ke SC*. 2004. Ammonia-induced structural changes of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy. Biochemistry 43, 10877-10885.
Fang CH, Hung CH, Chiang KA, Ke SC* and Chu HA*. 2005. Effect of ethylene glycol and methanol on ammonia-induced structural changes of the oxygen-evolving complex in photosystem II. Biochemistry 44, 9758-9765.
Hung CH, Huang JY, Chiu YF, and Chu HA*. 2007. "site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803." Biochim. Biophys. Acta, 1767, 686-693.
