Richard J. Debus

Rapid formation of the stable tyrosyl radical in photosystem II.

Two symmetrically positioned redox active tyrosine residues are present in the photosystem II (PSII) reaction center. One of them, TyrZ, is oxidized in the ns–μs time scale by P680+ and reduced rapidly (μs to ms) by electrons from the Mn complex. The other one, TyrD, is stable in its oxidized form and seems to play no direct role in enzyme function. Here, we have studied electron donation from these tyrosines to the chlorophyll cation (P680+) in Mn-depleted PSII from plants and cyanobacteria. In particular, a mutant lacking TyrZ was used to investigate electron donation from TyrD. By using EPR and time-resolved absorption spectroscopy, we show that reduced TyrD is capable of donating an electron to P680+ with t1/2 ≈ 190 ns at pH 8.5 in approximately half of the centers. This rate is ≈105 times faster than was previously thought and similar to the TyrZ donation rate in Mn-depleted wild-type PSII (pH 8.5). Some earlier arguments put forward to rationalize the supposedly slow electron donation from TyrD (compared with that from TyrZ) can be reassessed. At pH 6.5, TyrZ (t1/2 = 2–10 μs) donates much faster to P680+ than does TyrD (t1/2 > 150 μs). These different rates may reflect the different fates of the proton released from the respective tyrosines upon oxidation. The rapid rate of electron donation from TyrD requires at least partial localization of P680+ on the chlorophyll (PD2) that is located on the D2 side of the reaction center.

Evidence from FTIR Difference Spectroscopy of an Extensive Network of Hydrogen Bonds near the Oxygen-Evolving Mn4Ca Cluster of Photosystem II Involving D1-Glu65, D2-Glu312, and D1-Glu329

Analyses of the refined X-ray crystallographic structures of photosystem II (PSII) at 2.9-3.5 A have revealed the presence of possible channels for the removal of protons from the catalytic Mn(4)Ca cluster during the water-splitting reaction. As an initial attempt to verify these channels experimentally, the presence of a network of hydrogen bonds near the Mn(4)Ca cluster was probed with FTIR difference spectroscopy in a spectral region sensitive to the protonation states of carboxylate residues and, in particular, with a negative band at 1747 cm(-1) that is often observed in the S(2)-minus-S(1) FTIR difference spectrum of PSII from the cyanobacterium Synechocystis sp. PCC 6803. On the basis of its 4 cm(-1) downshift in D(2)O, this band was assigned to the carbonyl stretching vibration (C horizontal lineO) of a protonated carboxylate group whose pK(a) decreases during the S(1) to S(2) transition. The positive charge that forms on the Mn(4)Ca cluster during the S(1) to S(2) transition presumably causes structural perturbations that are transmitted to this carboxylate group via electrostatic interactions and/or an extended network of hydrogen bonds. In an attempt to identify the carboxylate group that gives rise to this band, the FTIR difference spectra of PSII core complexes from the mutants D1-Asp61Ala, D1-Glu65Ala, D1-Glu329Gln, and D2-Glu312Ala were examined. In the X-ray crystallographic models, these are the closest carboxylate residues to the Mn(4)Ca cluster that do not ligate Mn or Ca and all are highly conserved. The 1747 cm(-1) band is present in the S(2)-minus-S(1) FTIR difference spectrum of D1-Asp61Ala but absent from the corresponding spectra of D1-Glu65Ala, D2-Glu312Ala, and D1-Glu329Gln. The band is also sharply diminished in magnitude in the wild type when samples are maintained at a relative humidity of </=85%. It is proposed that D1-Glu65, D2-Glu312, and D1-Glu329 participate in a common network of hydrogen bonds that includes water molecules and the carboxylate group that gives rise to the 1747 cm(-1) band. It is further proposed that the mutation of any of these three residues, or partial dehydration caused by maintaining samples at a relative humidity of <or=85%, disrupts the network sufficiently that the structural perturbations associated with the S(1) to S(2) transition are no longer transmitted to the carboxylate group that gives rise to the 1747 cm(-1) band. Because D1-Glu329 is located approximately 20 A from D1-Glu65 and D2-Glu312, the postulated network of hydrogen bonds must extend for at least 20 A across the lumenal face of the Mn(4)Ca cluster. The D1-Asp61Ala, D1-Glu65Ala, and D2-Glu312Ala mutations also appear to substantially decrease the fraction of PSII reaction centers that undergo the S(3) to S(0) transition in response to a saturating flash. This behavior is consistent with D1-Asp61, D1-Glu65, and D2-Glu312 participating in a dominant proton egress channel that links the Mn(4)Ca cluster with the thylakoid lumen.

Degradation of the Photosystem II D1 and D2 proteins in different strains of the cyanobacterium Synechocystis PCC 6803 varying with respect to the type and level of psbA transcript

The turnover of the D1 and D2 proteins of Photosystem II (PSII) has been investigated by pulse-chase radiolabeling in several strains of the cyanobacterium Synechocystis PCC 6803 containing different types and levels of the psbA transcript. Strains lacking psbA1 and psbA3 gene and containing high levels of the psbA2 transcript showed the selective synthesis of D1 whose degradation could be slowed down by the protein synthesis inhibitor lincomycin. In contrast, in strains containing just the psbA3 gene, the intensity of the D1 protein labeling was lower and labeling of the D2 and CP43 proteins was stimulated in comparison to the psbA2-containing strains. In addition, the rate and selectivity of the D1 degradation and its dependence on the presence of lincomycin was proportional to the level of the psbA3 transcript in the particular strain. Consequently, there was parallel, lincomycin-independent and slowed-down breakdown of the D1 and D2 proteins in strains with the lowest level of psbA3 transcript. These results are discussed in terms of a model in which the rate of D1 and D2 degradation in cyanobacteria is affected not only by the rate of PSII photodamage, but also by the availability of newly synthesized D1 protein. Moreover, the comparison of the non-oxygen-evolving D1 mutants D170A** and Y161F*** differing by the presence of tyrosine Z has indicated a minor role of the oxidized form of this secondary PSII electron donor in the donor side mechanism of D1 and D2 protein breakdown.

FTIR studies of metal ligands, networks of hydrogen bonds, and water molecules near the active site Mn4CaO5 cluster in Photosystem II

The photosynthetic conversion of water to molecular oxygen is catalyzed by the Mn₄CaO₅ cluster in Photosystem II and provides nearly our entire supply of atmospheric oxygen. The Mn₄CaO₅ cluster accumulates oxidizing equivalents in response to light-driven photochemical events within Photosystem II and then oxidizes two molecules of water to oxygen. The Mn₄CaO₅ cluster converts water to oxygen much more efficiently than any synthetic catalyst because its protein environment carefully controls the clusters reactivity at each step in its catalytic cycle. This control is achieved by precise choreography of the proton and electron transfer reactions associated with water oxidation and by careful management of substrate (water) access and proton egress. This review describes the FTIR studies undertaken over the past two decades to identify the amino acid residues that are responsible for this control and to determine the role of each. In particular, this review describes the FTIR studies undertaken to characterize the influence of the clusters metal ligands on its activity, to delineate the proton egress pathways that link the Mn₄CaO₅ cluster with the thylakoid lumen, and to characterize the influence of specific residues on the water molecules that serve as substrate or as participants in the networks of hydrogen bonds that make up the water access and proton egress pathways. This information will improve our understanding of water oxidation by the Mn₄CaO₅ catalyst in Photosystem II and will provide insight into the design of new generations of synthetic catalysts that convert sunlight into useful forms of storable energy. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

Mutation of lysine 317 in the D2 subunit of photosystem II alters chloride binding and proton transport.

The role of chloride in photosystem II (PSII) is unclear. Using structural information from PSII and a careful comparison with other chloride-activated enzymes, we proposed a role for chloride at the D2-K317 site in PSII [Pokhrel, R., et al. (2011) Biochemistry 50, 2725-2734]. To probe the role of chloride at this site, the D2-K317R, D2-K317A, D2-K317Q, and D2-K317E mutations were created in the cyanobacterium Synechocystis sp. PCC 6803. Purified PSII from the mutants was probed with Fourier transform infrared difference spectroscopy, demonstrating that compared to PSII from wild-type Synechocystis, PSII from all four mutants exhibit changes in the conformations of the polypeptide backbone and carboxylate groups. However, D2-K317R PSII exhibits minor changes, whereas D2-K317A, D2-K317Q, and D2-K317E PSII exhibit more substantial changes in polypeptide conformations. Steady-state oxygen-evolution measurements of purified PSII core complexes show that the oxygen-evolution activity of D2-K317A is independent of chloride. This is consistent with the loss of the chloride requirement when the charged K residue is replaced with an uncharged residue that no longer binds to an essential carboxylate (D1-D61) in the absence of chloride, analogous to observations in other chloride-activated enzymes. In contrast, the oxygen-evolution activity of D2-K317R is sensitive to the chloride concentration in the assay buffer; the effective KD for chloride binding is higher in D2-K317R than in wild-type PSII, possibly because of a less optimal binding site in the mutant. The S2 states of wild-type, D2-K317A, and D2-K317R PSII were probed using electron paramagnetic resonance spectroscopy. A g = 2 multiline signal, similar to the wild-type signal, was observed for D2-K317A and D2-K317R. However, a g = 4 signal was also observed for D2-K317R. Measurements of flash-dependent O2 yields showed that D2-K317A and D2-K317R have a higher miss factor than wild-type PSII. The oxygen-release kinetics of D2-K317A and D2-K317R were slower than those of the wild type, in the following order: D2-K317A < D2-K317R < wild type. These results collectively suggest that proton transfer is inefficient in D2-K317A and D2-K317R, thereby giving rise to a higher miss factor and slower oxygen-release kinetics.

Ligation of D1-His332 and D1-Asp170 to the Manganese Cluster of Photosystem II from Synechocystis Assessed by Multifrequency Pulse EPR Spectroscopy

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is used to ascertain the nature of the bonding interactions of various active site amino acids with the Mn ions that compose the oxygen-evolving cluster (OEC) in photosystem II (PSII) from the cyanobacterium Synechocystis sp. PCC 6803 poised in the S(2) state. Spectra of natural isotopic abundance PSII ((14)N-PSII), uniformly (15)N-labeled PSII ((15)N-PSII), and (15)N-PSII containing (14)N-histidine ((14)N-His/(15)N-PSII) are compared. These complementary data sets allow for a precise determination of the spin Hamiltonian parameters of the postulated histidine nitrogen interaction with the Mn ions of the OEC. These results are compared to those from a similar study on PSII isolated from spinach. Upon mutation of His332 of the D1 polypeptide to a glutamate residue, all isotopically sensitive spectral features vanish. Additional K(a)- and Q-band ESEEM experiments on the D1-D170H site-directed mutant give no indication of new (14)N-based interactions.

Ammonia Binds to the Dangler Manganese of the Photosystem II Oxygen-Evolving Complex

High-resolution X-ray structures of photosystem II reveal several potential substrate binding sites at the water-oxidizing/oxygen-evolving 4MnCa cluster. Aspartate-61 of the D1 protein hydrogen bonds with one such water (W1), which is bound to the dangler Mn4A of the oxygen-evolving complex. Comparison of pulse EPR spectra of (14)NH3 and (15)NH3 bound to wild-type Synechocystis PSII and a D1-D61A mutant lacking this hydrogen-bonding interaction demonstrates that ammonia binds as a terminal NH3 at this dangler Mn4A site and not as a partially deprotonated bridge between two metal centers. The implications of this finding on identifying the binding sites of the substrate and the subsequent mechanism of dioxygen formation are discussed.

Evidence from FTIR difference spectroscopy that D1-Asp61 influences the water reactions of the oxygen-evolving Mn4CaO5 cluster of photosystem II.

Understanding the mechanism of photosynthetic water oxidation requires characterizing the reactions of the water molecules that serve as substrate or that otherwise interact with the oxygen-evolving Mn4CaO5 cluster. FTIR difference spectroscopy is a powerful tool for studying the structural changes of hydrogen bonded water molecules. For example, the O-H stretching mode of water molecules having relatively weak hydrogen bonds can be monitored near 3600 cm(-1), the D-O-D bending mode can be monitored near 1210 cm(-1), and highly polarizable networks of hydrogen bonds can be monitored as broad features between 3000 and 2000 cm(-1). The two former regions are practically devoid of overlapping vibrational modes from the protein. In Photosystem II, water oxidation requires a precisely choreographed sequence of proton and electron transfer steps in which proton release is required to prevent the redox potential of the Mn4CaO5 cluster from rising to levels that would prevent its subsequent oxidation. Proton release takes place via one or more proton egress pathways leading from the Mn4CaO5 cluster to the thylakoid lumen. There is growing evidence that D1-D61 is the initial residue of one dominant proton egress pathway. This residue interacts directly with water molecules in the first and second coordination spheres of the Mn4CaO5 cluster. In this study, we explore the influence of D1-D61 on the water reactions accompanying oxygen production by characterizing the FTIR properties of the D1-D61A mutant of the cyanobacterium, Synechocystis sp. PCC 6803. On the basis of mutation-induced changes to the carbonyl stretching region near 1747 cm(-1), we conclude that D1-D61 participates in the same extensive networks of hydrogen bonds that have been identified previously by FTIR studies. On the basis of mutation-induced changes to the weakly hydrogen-bonded O-H stretching region, we conclude that D1-D61 interacts with water molecules that are located near the Cl(-)(1) ion and that deprotonate or participate in stronger hydrogen bonds as a result of the S1 to S2 and S2 to S3 transitions. On the basis of the elimination of a broad feature between 3100 and 2600 cm(-1), we conclude that the highly polarizable network of hydrogen bonds whose polarizability or protonation state increases during the S1 to S2 transition involves D1-D61. On the basis of the elimination of features in the D-O-D bending region, we conclude that D1-D61 forms a hydrogen bond to one of the H2O molecules whose H-O-H bending mode changes in response to the S1 to S2 transition. The elimination of this H2O molecule in the D1-D61A mutant provides one rationale for the decreased efficiency of water oxidation in this mutant. Finally, we discuss reasons why the recent conclusion that a substrate-containing cluster of five water molecules accepts a proton from the Mn4CaO5 cluster during the S1 to S2 transition and deprotonates during subsequent S state transitions should be reassessed.

Network of hydrogen bonds near the oxygen-evolving Mn(4)CaO(5) cluster of photosystem II probed with FTIR difference spectroscopy.

We previously provided experimental evidence that an extensive network of hydrogen bonds exists near the oxygen-evolving Mn4CaO5 cluster in photosystem II and that elements of this network form part of a dominant proton-egress pathway leading from the Mn4CaO5 cluster to the thylakoid lumen. The evidence was based on (i) the elimination of the same ν(C═O) mode of a protonated carboxylate group in the S2-minus-S1 FTIR difference spectrum of wild-type PSII core complexes from the cyanobacterium Synechocystis sp. PCC 6803 by the mutations D1-E65A, D2-E312A, and D1-E329Q and (ii) the substantial decrease in the efficiency of the S3 to S0 transition caused by the mutations D1-D61A, D1-E65A, and D2-E312A. The eliminated ν(C═O) mode corresponds to an unidentified carboxylate group whose pKa value decreases in response to the increased charge that develops on the Mn4CaO5 cluster during the S1 to S2 transition. In the current study, we have extended our work to include the ν(C═O) regions of other Sn+1-minus-Sn FTIR difference spectra and to additional mutations of residues inferred to participate in networks of hydrogen bonds near the Mn4CaO5 cluster or leading from the Mn4CaO5 cluster to the thylakoid lumen. Our data suggest that a different carboxylate group has its pKa value increased during the S2 to S3 transition and that a third carboxylate group experiences a change in its environment during the S0 to S1 transition. The pKa values that shift during the S1 to S2 and S2 to S3 transitions appear to be restored during the S3 to S0 transition. The D1-R334A mutation decreases or eliminates the same ν(C═O) modes from the S2-minus-S1 and S3-minus-S2 spectra as mutations D1-E65A, D2-E312A, and D1-E329Q and substantially decreases the efficiency of the S3 to S0 transition. We conclude that D1-R334 participates in the same dominant proton-egress pathway that was identified in our previous study. The D1-Q165E mutation leaves the ν(C═O) region of the S2-minus-S1 FTIR difference spectrum intact, but it eliminates a mode from this region of the S3-minus-S2 spectrum. We conclude that D1-Q165 participates in an extensive network of hydrogen bonds that that extends across the Mn4CaO5 cluster to the D1-E65/D2-E312 dyad and that includes D1-E329 and several water molecules including the W2 and W3 water ligands of the Mn4CaO5 clusters dangling MnA4 and Ca ions, respectively. The D2-E307Q, D2-D308N, D2-E310Q, and D2-E323Q mutations alter the ν(C═O) regions of none of the FTIR difference spectra. We conclude that these four residues are located far from the three unidentified carboxylate groups that give rise to the ν(C═O) features observed in the FTIR difference spectra.

13C ENDOR Reveals That the D1 Polypeptide C-Terminus Is Directly Bound to Mn in the Photosystem II Oxygen Evolving Complex

Antiferromagnetically coupled Mn(III)Mn(IV) dimers have been commonly used to study biological systems that exhibit complex exchange interactions. Such is the case for the oxygen evolving complex (OEC) in photosystem II (PSII), where we have studied whether the C-terminal carboxylate of D1-Ala344 is directly bound to the Mn cluster. To probe these protein-derived carboxylate hyperfine interactions, which give direct bonding information, Q-band (34 GHz) Mims ENDOR was performed on a Mn(III)Mn(IV) dimer ([Mn(III)Mn(IV)(mu-O)(2)mu-OAc(TACN)(2)](BPh(4))(2)) (1) that was labeled with (13)C (I = (1)/(2)) at the carboxylate position of the acetate bridge. A(dip) is computed based on atomic coordinates from available X-ray crystal structures to be [-2.4, -0.8, 3.2] MHz. The value for A(iso) was determined based on simulation of the experimental ENDOR data, for complex 1 A(iso) = -1 MHz. Similar studies were then performed on PSII from Synechocystis sp. PCC 6803, in which all alanine-derived C=O groups are labeled with (13)C including the C-terminal alpha-COO(-) group of D1 (Ala344), as well as PSII proteins uniformly labeled with (13)C. Using recent X-ray crystallography data from T. elongatus the values for A(dip) were calculated and simulations of the experimental data led to A(iso) values of 1.2, 1, and 2 MHz, respectively. We infer from complex 1 that an A(iso) significantly larger than 1.2 MHz for a Mn-coordinating carboxylate moiety is unlikely. Therefore, we support the closer arrangement of Ala344 suggested by the Loll and Guskov structures and conclude that the C-terminal carboxylate of D1 polypeptide is directly bound to the Mn cluster.