Bruce A. Diner

Amino acid residues involved in the coordination and assembly of the manganese cluster of photosystem II. Proton-coupled electron transport of the redox-active tyrosines and its relationship to water oxidation.

The combination of site-directed mutagenesis, isotopic labeling, new magnetic resonance techniques and optical spectroscopic methods have provided new insights into cofactor coordination and into the mechanism of electron transport and proton-coupled electron transport in photosystem II. Site-directed mutations in the D1 polypeptide of this photosystem have implicated a number of histidine and carboxylate residues in the coordination and assembly of the manganese cluster, responsible for photosynthetic water oxidation. Many of these are located in the carboxy-terminal region of this polypeptide close to the processing site involved in its maturation. This maturation is a required precondition for cluster assembly. Recent proposals for the mechanism of water oxidation have directly implicated redox-active tyrosine Y(Z) in this mechanism and have emphasized the importance of the coupling of proton and electron transfer in the reduction of Y(Z)(radical) by the Mn cluster. The interaction of both homologous redox-active tyrosines Y(Z) and Y(D) with their respective homologous proton acceptors is discussed in an effort to better understand the significance of such coupling.

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.

Identification of Q400, a high-potential electron acceptor of Photosystem II, with the iron of the quinone-iron acceptor complex

Measurements of the area bounded by the variable fluorescence induction curve and the maximum fluorescence yield as a function of redox potential led I. Ikegami and S. Katoh ((1973) Plant Cell Physiol. 14, 829–836) to propose the existence of a high-potential electron acceptor, Q400 (Em7.8 = 360 mV), associated with Photosystem II (PS II). We have generated the oxidized form of this acceptor (Q+400) using ferricyanide and other oxidants in thylakoid membranes isolated from a mutant of Chlamydomonas reinhardtii lacking Photosystem I and the cytochrome b6f complex. Q+400 was detected by a decrease in the extent of reduction of the primary quinone electron acceptor, QA, in a low-intensity light flash exciting PS II reaction centers only once. EPR measurements in the presence of Q+400 indicated the presence of new signals at g = 8, 6.4 and 5.5. These disappeared upon illumination at 200 K or upon reduction with ascorbate. Mossbauer absorption attributed to the Fe2+ of the QA-Fe2+ acceptor complex of PS II disappeared upon addition of ferricyanide due to the formation of Fe3+. The Fe2+ signal was restored by subsequent addition of ascorbate. All of these spectroscopic signals show similar pH-dependent (n = 1) midpoint potentials (approx. −60 mV / pH unit) and an Em7.5 = 370 mV. We assign the EPR signals to the Fe3+ state of the quinone-iron acceptor. Electron transfer to the Fe3+ is responsible for the decrease in QA reduction upon single-hit flash excitation. The properties of the Fe3+Fe2+ redox couple are consistent with those of Q+400Q400 and we conclude that the iron of the QA-Fe acceptor complex is responsible for this species.

Dependence of the deactivation reactions of photosystem II on the redox state of plastoquinone pool A varied under anaerobic conditions; Equilibria on the acceptor side of photosystem II.

Dark adapted spinach chloroplasts and Chlorella with variably reduced plastoquinone pools were given 1 or 2 saturating flashes. Under these conditions the rate of deactivation of state S2 of the oxygen evolving site of Photosystem II (B, Kok, B, Forbush, M. McGloin (1970) Photochem. Photobiol. 11, 457-475) is highly dependent on the pool redox state, undergoing a nearly 10-fold acceleration upon transforming the plastoquinone pool (A) from 100% oxidized to 90% reduced. Deactivation of state S3 is unaffected by the same variation of the pool redox state. These observations are attributed to a high concentration of Photosystem II reduced primary electron acceptor, Q-, coincident with the formation of S2 and a low concentration coincident with the formation of S3, under the conditions of highly reduced plastoquinone pool. Simultaneous determination of Q- and A2- result in an estimated equilibrium constant of 15-20 for reaction Q-B in equilibrium QB- and a value greater than 50 for equilibrium Q-B- in equilibrium QB2-, where B is the secondary electron acceptor described by B. Bouges-Bocquet ((1973) Biochim. Biophys. Acta. 314, 250-256) and B.R. Velthuys and J. Amesz ((1974) Biochim. Biophys. Acta. 333, 85-94). It is proposed that doubly reduced B becomes protonated in the last reaction.

Direct detection of the electron acceptor of photosystem II: Evidence that Q is an iron—quinone complex

The primary pboto~h~~st~ of the photosystem II reaction centre involves the transfer of an electron from the primary chlorophyll donor (P680) to the primary acceptor, Q. The properties of Q, a specialised plastoquinone [ 1,2] have been studied in detail using indirect fluorescence measurements [3-61 and absorbance changes [7-l 21 but no electron paramagnetic resonance (EPR) signal was observed to confirm the suggestion [ 10,l l] that as in bacterial reaction centres, a transition metal ion (probably Fe’+) was complexed to Q. When Q is chemic~y reduced, the photoredu~tion of a pheophytin intermediate electron carrier (I) has been observed [ 131. Recent EPR investigations have identified a signal near g = 2.00 corresponding to I[14-171 which, when present in samples containing Q-, gives rise to an EPR doublet [ 14,151 with similar properties to those found in purple photosynthetic bacterial reaction centres (reviewed in [IS]). This and further work [ 191 strongly suggested that the interaction producing the doublet involved a semiquinoneiron complex of the type exhibiting characteristic EPR signals near g = 1.82 in purple photosynthetic bacteria [20-231. Using broken c~oroplasts from a mutant of barley (Hordeurn vulgare viridis zb 63)lacking photosystem I [16,17], the photoreduction of Iwas also observed at 5-77 K indicating the presence of a fast donor to P680 under these conditions. In 1241 a highly active photosystem II particle was prepared from a mutant of the green alga ~~~ydornonas reinhardii, originally isolated by P. Bennoun. Fluorescence measurements [24] suggest that the particles lack the secondary quinone acceptor B. Using

Crystal structures of the photosystem II D1 C-terminal processing protease.

We report here the first three-dimensional structure of the D1 C-terminal processing protease (D1P), which is encoded by the ctpA gene. This enzyme removes the C-terminal extension of the D1 polypeptide of photosystem II of oxygenic photosynthesis. Proteolytic processing is necessary to allow the light driven assembly of the tetranuclear manganese cluster, which is responsible for photosynthetic water oxidation. The X-ray structure of the Scenedesmus obliquus enzyme has been determined at 1.8 Å resolution using the multiwavelength anomalous dispersion method. The enzyme is monomeric and is composed of three folding domains. The middle domain is topologically homologous to known PDZ motifs and is proposed to be the site at which the substrate C-terminus binds. The remainder of the substrate likely extends across the face of the enzyme, interacting at its scissile bond with the enzyme active site Ser 372 / Lys 397 catalytic dyad, which lies at the center of the protein at the interface of the three domains.

Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. II. Evidence that HCO−3/CO2 binds to the acceptor-side non-heme iron

We have demonstrated (Petrouleas and Diner, accompanying paper) that NO binds to the non-heme iron of the PS II reaction center. We show here that, in spinach chloroplasts, NO (Kd ≈ 30 μM), like formate, slows electron transfer between the primary and secondary quinone electron acceptors of PS II, QA and QB, respectively. In a series of saturating flashes given to dark-adapted chloroplasts treated with NO, this electron transfer is slowed by at least a factor of 10 from the second saturating flash excitation onward as compared to untreated chloroplasts. This slowing is completely reversed by the addition of NaHCO3 (10 mM), indicating that NO, like formate, displaces bicarbonate from the reaction center. The NO-enhanced dissociation of HCO−3 from the reaction center is pH-dependent, occurring much faster at pH 5.5 than at 7.4. In the reverse experiment, the S = 32 Fe(II)-NO EPR signal at g = 4 is diminished by the addition of NaHCO3, indicating that HCO−3 dissociates the NO ligation to the iron. These data argue in favor of HCO−3/CO2 as a ligand to the iron. Formate does not dissociate NO from the iron and it is possible that formate and NO displace HCO−3/CO2 by different mechanisms.

Spectroscopic Properties of Reaction Center Pigments in Photosystem II Core Complexes: Revision of the Multimer Model

Absorbance difference spectra associated with the light-induced formation of functional states in photosystem II core complexes from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 (e.g., P(+)Pheo(-),P(+)Q(A)(-),(3)P) are described quantitatively in the framework of exciton theory. In addition, effects are analyzed of site-directed mutations of D1-His(198), the axial ligand of the special-pair chlorophyll P(D1), and D1-Thr(179), an amino-acid residue nearest to the accessory chlorophyll Chl(D1), on the spectral properties of the reaction center pigments. Using pigment transition energies (site energies) determined previously from independent experiments on D1-D2-cytb559 complexes, good agreement between calculated and experimental spectra is obtained. The only difference in site energies of the reaction center pigments in D1-D2-cytb559 and photosystem II core complexes concerns Chl(D1). Compared to isolated reaction centers, the site energy of Chl(D1) is red-shifted by 4 nm and less inhomogeneously distributed in core complexes. The site energies cause primary electron transfer at cryogenic temperatures to be initiated by an excited state that is strongly localized on Chl(D1) rather than from a delocalized state as assumed in the previously described multimer model. This result is consistent with earlier experimental data on special-pair mutants and with our previous calculations on D1-D2-cytb559 complexes. The calculations show that at 5 K the lowest excited state of the reaction center is lower by approximately 10 nm than the low-energy exciton state of the two special-pair chlorophylls P(D1) and P(D2) which form an excitonic dimer. The experimental temperature dependence of the wild-type difference spectra can only be understood in this model if temperature-dependent site energies are assumed for Chl(D1) and P(D1), reducing the above energy gap from 10 to 6 nm upon increasing the temperature from 5 to 300 K. At physiological temperature, there are considerable contributions from all pigments to the equilibrated excited state P*. The contribution of Chl(D1) is twice that of P(D1) at ambient temperature, making it likely that the primary charge separation will be initiated by Chl(D1) under these conditions. The calculations of absorbance difference spectra provide independent evidence that after primary electron transfer the hole stabilizes at P(D1), and that the physiologically dangerous charge recombination triplets, which may form under light stress, equilibrate between Chl(D1) and P(D1).

Light-induced oxidation of the acceptor-side Fe(II) of Photosystem II by exogenous quinones acting through the QB binding site. I. Quinones, kinetics and pH-dependence

Abstract We have recently shown by optical, EPR and Mossbauer spectroscopy that the high spin Fe(II) of the quinone-iron acceptor complex of Photosystem II can be oxidized by ferricyanide to high-spin Fe(III). The midpoint potential of the Fe(III)/Fe(II) couple is 370 mV at pH 7.5 and shows an approximate pH-dependence of −60 mV/pH unit. The iron was identified as being responsible for the high potential Photosystem II acceptor known as Q400, discovered by Ikegami and Katoh ((1975) Plant Cell Physiol. 14, 829–836) but until now not identified chemically. We establish here that QA and the oxidized Fe(III) are linked in series, with QA the first to be reduced in the primary charge separation of Photosystem II. At pH 7.5, an electron is then transferred from Q−A to Fe(III) with a t 1 2 of 25 μs, reforming QA Fe(II). The Fe(II) can also be oxidized to Fe(III) in oxygen-evolving thylakoid membranes through a photoreduction-induced oxidation in the presence of exogenous quinones, where E m ,7 ( Q − QH 2 ) > E m ,7 ( Fe(III) Fe(II) ) − 60 mV . Single turnover illumination of the Photosystem II reaction center at 200 K, followed by warming to 0°C, results in photoreduction of these quinones to the semiquinone form which in turn oxidizes the Fe(II) to Fe(III). A second turnover of the reaction center reduces Fe(III) back to Fe(II). These reactions, similar to those reported by Zimmermann and Rutherford (Zimmermann, J.L. and Rutherford, A.W. (1986) Biochim. Biophys. Acta 851, 416–423) at room temperature, in work largely done in parallel, are summarized below: where QA and Qex are the primary quinone acceptor of Photosystem II and exogenous quinone, respectively. Detection of Fe(III) at g = 8 by EPR spectroscopy shows this signal to oscillate with period two upon successive turnovers of the Photosystem II reaction center. Different exogenous quinones give different EPR spectra for Fe(III), indicating that these bind close to the Fe binding site and modify the symmetry of the Fe(III) environment. A study of the pH-dependence of the light-induced oxidation of the Fe(III) by phenyl-p-BQ shows a pH-optimum at 6–7. The decline at higher pH is consistent with a pH-dependence of −60 mV/pH unit and −120 mV/pH unit, respectively, for redox couples Fe(III)/Fe(II) and Q−/QH2. The decline at lower pH was not foreseen and appears associated with a transformation of the quinone-iron environment from that showing a Q−AFe(II) EPR resonance of g = 1.9 at high pH to one at g = 1.84 below pH 6.5. The latter form appears not to support light-induced oxidation of the Fe(II) by exogenous quinones.

Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. I. NO binds to the acceptor-side non-heme iron

NO is a good electrophile and EPR spin probe, carrying an unpaired electron (S = 12). Exposure of Photosystem II reaction centers to NO results in the appearance of an EPR signal at g = 4. Dark titration with NO shows a Kd ≈ 30 μM in spinach chloroplasts and 250 μM in BBY preparations. Successive cycles of illumination at 200 K, followed by incubation at 245 K, results in binary oscillations of the amplitude of the g = 4 signal in spinach chloroplasts. This signal is small in states Q−AQB, Q−AQ−B, but large in states QAQB and QAQBH2 of the PS II acceptor side. NO slows electron transfer between QA and QB (Diner, B.A and Petrouleas, V. (1990) Biochim. Biophys. Acta 1015, 141–149) and modifies the Mossbauer spectrum of the non-heme Fe(II). These results strongly support the assignment of the g = 4 EPR signal to an acceptor side Fe(II)-NO adduct in an S = 32 state. Exchange coupling of this species with the S = 12 semiquinones results in an integral spin system, not detectable in X-band EPR. The g = 4 spectrum can be described by a spin hamiltonian. The rhombicity parameter ED is in all cases small (⩽ 0.015), implying a near axial environment. NO can donate electrons to PS II. Charge recombination, following a light flash in the presence of DCMU, is blocked by NO (Km ≈ 30 μM, 25°C). NO also reacts reversibly in the dark with D+ (an oxidized secondary donor), resulting in the disappearance of Signal IIdark with a Kd of 3 μM. Pumping off of NO in the dark results in full recovery of the signal.