Nikos Lydakis-Simantiris

A hydrogen-atom abstraction model for the function of YZ in photosynthetic oxygen evolution

Recent magnetic-resonance work on YŻ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YŻ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.

Manganese and tyrosyl radical function in photosynthetic oxygen evolution

Photosystem II catalyzes the photosynthetic oxidation of water to O2. The structural and functional basis for this remarkable process is emerging. The catalytic site contains a tetramanganese cluster, calcium, chloride and a redox-active tyrosine organized so as to promote electroneutral hydrogen atom abstraction from manganese-bound substrate water by the tyrosyl radical. Recent work is assessed within the framework of this model for the water oxidizing process.

Kinetic isotope effects on the reduction of the Yz radical in oxygen evolving and tris-washed photosystem II membranes by time-resolved EPR

Abstract Deuterium kinetic isotope effects on the electron transfer reactions for the reduction of the tyrosyl radical Y ⋅ Z were studied with time resolved electron paramagnetic resonance in oxygen evolving N and tris-washed photosystem II membranes. For the electron transfers from the (Mn) 4 /H 2 O complex to Y ⋅ Z , S-state dependent kinetic isotope effects were observed with k H / k D values of 2.9, 1.3 and 1.6 for the transitions S 1 →S 2 , S 2 →S 3 and S 3 →[S 4 ]→S 0 , respectively. In tris-washed samples, oxygen evolution is abolished and the reduction of Y ⋅ Z proceeds at the same rate in 1 H 2 O and in 2 H 2 O. Taken together, these results show that proton-electron coupling during the reduction of Y ⋅ Z is in effect when water is the electron donor to PSII, but not when oxygen evolution is inhibited. This conclusion is consistent with a mechanism for oxygen evolution in which Y ⋅ Z abstracts a hydrogen atom from the (Mn) 4 /H 2 O complex on each S-state transition. By demonstrating proton/electron coupling, our data suggest that these hydrogen atom abstraction reactions proceed along a pathway that has a concerted character. The atom transfer character in the transition state serves to minimize charge development during the S n Y ⋅ Z →S n +1 Y Z reactions and is consistent with the relatively low activation energies observed in the S-state transitions in PSII.

Biochemical and Spectroscopic Properties of Heat-Treated Manganese Stabilizing Protein

Three water soluble proteins with molecular masses of 17, 23 and 33 kDa, located on the lumenal side of photosystem 2 (PSII), are responsible for the stability and the optimum function of the inorganic cofactors of the oxygen evolving complex (OEC) (1). More specifically, 17 and 23 kDa proteins are responsible for retention of Ca2+ and Cl− whereas a 33 kDa protein, called Manganese Stabilizing Protein (MSP), is responsible for the integrity and optimum function of the manganese cluster of OEC (1,2). Protein digestion and cross linking studies have identified the E loops of the intrinsic, chlorophyll-a binding proteins CP47 and CP43 as binding sites of MSP (3–5). Cyt b559 and a small intrinsic protein (<10 kDa) have also been proposed to provide ligands for the protein (5,6). Biochemical studies have shown that there are two copies of MSP per PSII reaction center (7,8). Protein digestion and site-directed mutagenesis have identified the N- terminus and the C- terminus of MSP as responsible for binding to CP47 and for maintaining structural features in solution necessary for functional binding, respectively (9, Betts et al, unpublished results). Here we report the biochemical and spectroscopic properties of native and recombinant MSP incubated at either room temperature or at 90°C. We show that heated MSP retains a remarkable ability to rebind to PSII and reactivate O2 evolution after long incubation at high temperatures. The secondary and tertiary structures of both species were examined by far-UV circular dichroism (CD), near-UV CD and size-exclusion chromatography. Cooling of heat-treated MSP results in refolding to a nearly native conformation. Structural and functional thermostability, along with other properties of MSP, suggest that it belongs to a family of “natively unfolded” proteins that participate in protein-protein interactions (10).

Bacterial Overexpression and Site-directed Mutagenesis of the PS2 Manganese Stabilizing Protein: Progress in Elucidation of Structure and Function

Manganese Stabilizing Protein (MSP) was discovered and purified by Kuwabara and Murata (1), and has since proven to be an essential extrinsic component of PS2 (reviewed in 2,3). Extraction of MSP modifies the tetranuclear Mn cluster of the O2-evolving complex; 2 Mn are released as Mn2+, the S2 and S3 states are abnormally stable, and the S3 → S4 → S0 step is slowed by a factor of 3–5. The isolated spinach protein is comprised of 247 amino acids with a molecular mass of 26,535 and a pi of 5.2. Analyses of secondary structure by CD shows that the protein in solution is comprised of α-helix (10%), β-sheet (33–38%) and about 50% turns and random coil (4,5). The technique of site-directed mutagenesis is a useful tool for probing protein structure and function. For MSP, two approaches have been applied. In the first, mutagenesis in vivo has been utilized in cyanobacteria (6). In the second, a method first used by Seidler and Michel (7) for overexpression and processing of precursor eukaryotic proteins in E. coli has provided a means for mutagenesis of MSP that is facilitated by the ease with which the overexpressed protein can be rebound to MSP-depleted PS2 samples. We have modified this method so that MSP inclusion bodies can be harvested from E. coil purified, and used to reconstitute high levels of activity (8). Here, we report on the use of recombinant wildtype and mutant forms of MSP in experiments designed to characterize the structure and function of this important PS2 protein.