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Metal complexes are extensively explored as catalysts for oxidation reactions; molecular-based mechanisms are usually proposed for such reactions. However, the roles of the decomposition products of these materials in the catalytic process have yet to be considered for these reactions. Herein, the cyclohexene oxidation in the presence of manganese(III) 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride) (1) in a heterogeneous system via loading the complex on an SBA-15 substrate is performed as a study case. A molecular-based mechanism is usually suggested for such a metal complex. Herein, 1 was selected and investigated under the oxidation reaction by iodosylbenzene or (diacetoxyiodo)benzene (PhI(OAc)(2)). In addition to 1, at least one of the decomposition products of 1 formed during the oxidation reaction could be considered a candidate to catalyze the reaction. First-principles calculations show that Mn dissolution is energetically feasible in the presence of iodosylbenzene and trace amounts of water. Metal complexes are often used as catalysts for oxidation reactions, however, there are open questions about the role of the decomposition products in the catalytic process. Here, the authors explore the potential role of decomposition products in the oxidation of cyclohexene using a manganese(III) complex catalyst adsorbed on an SBA-15 substrate.

After a brief background on Otto Heinrich Warburg (1883–1970), and some of his selected research, we provide highlights, in English, of three of his papers in the 1940s—unknown to many as they were not originally published in English. They are: two brief reports on Photosynthesis, with Wilhelm Luttgens, originally published in German, in 1944: ‘Experiment on assimilation of carbonic acid’; and ‘Further experiments on carbon dioxide assimilation’. This is followed by a regular paper, originally published in Russian, in 1946: ‘The photochemical reduction of quinone in green granules’. Since the 1944 reports discussed here are very short, their translations are included in the Appendix, but that of the 1946 paper is provided in the Supplementary Material. In all three reports, Warburg provides the first evidence for and elaborates on light-driven water oxidation coupled to reduction of added benzoquinone. These largely overlooked studies of Warburg are in stark contrast to Warburg’s well-known error in assigning the origin of the photosynthetically formed dioxygen to carbonate.

In soluble methane monooxygenase enzymes (sMMO), dioxygen (O2) is activated at a diiron(II) center to form an oxodiiron(IV) intermediate Q that performs the challenging oxidation of methane to meth...

Information on binding and rearrangement of pivotal water molecules could support understanding of light-driven water oxidation at the catalytic Mn4CaO5 cluster of photosystem II (PSII). To address this point, the binding of ammonia (NH3)—a possible substrate-water analogue—has been investigated and discussed in the context of putative reaction mechanisms. By time-resolved detection of O2 formation after light-flash excitation, we discriminate three NH3/NH4+ binding sites jointly characterized by a Km value around 25 mM (of NH4+), but differing in their influence on the O2-formation step. At 100 mM NH4Cl (pH 7.5), we observe (1) a PSII fraction with complete inhibition of O2-formation, (2) fast O2-formation with a time constant of 1.7 ms at 20 °C (Fast-PSII), and (3) slow O2-formation with a time constant of 36 ms at 20 °C (Slow-PSII). For the Fast-PSII, we determine an activation enthalpy of 223 ± 11 meV. Activation enthalpy and entropy of the Fast-PSII are essentially identical to the corresponding figures in the absence NH3/NH4+ binding. For the Slow-PSII, the activation enthalpy is 323 ± 11 meV and thus significantly increased, whereas the activation entropy remains essentially unchanged. We conclude: (1) The fully-inhibitory binding site could relate to bound NH3 replacing one of the two substrate-water molecules. (2) The Fast-PSII may relate to NH3/NH4+ binding in the S2-state of PSII followed by unbinding before onset of the O O bond formation step, but also more intricate mechanisms are not excluded. (3) In the Slow-PSII, NH3/NH4+ binding increases the energetic barrier of the O O bond formation step significantly.

Understanding the mechanism for electrochemical water oxidation is important for the development of more efficient catalysts for artificial photosynthesis. A basic step is the proton-coupled electron transfer, which enables accumulation of oxidizing equivalents without buildup of a charge. We find that substituting deuterium for hydrogen resulted in an 87% decrease in the catalytic activity for water oxidation on Co-based amorphous-oxide catalysts at neutral pH, while

A rate enhancement of one to two orders of magnitude can be obtained in the aldehyde deformylation reactions by replacing the –N(CH3) groups of [NiIII(O2)(Me4[12]aneN4)]+ (Me4[12]aneN4 = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane) and [NiIII(O2)(Me4[13]aneN4)]+ (Me4[13]aneN4 = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclotridecane) complexes by –NH in [NiIII(O2)([12]aneN4)]+ (2; [12]aneN4 = 1,4,7,10-tetraazacyclododecane) and [NiIII(O2)([13]aneN4)]+ (4; [13]aneN4 = 1,4,7,10-tetraazacyclotridecane). Based on detailed spectroscopic, reaction-kinetics and theoretical investigations, the higher reactivities of 2 and 4 are attributed to the changes in the secondary-sphere interactions between the [NiIII(O2)]+ and [12]aneN4 or [13]aneN4 moieties, which open up an alternative electrophilic pathway for the aldehyde oxidation reaction. Identification of primary kinetic isotope effects on the reactivity and stability of 2 when the –NH groups of the [12]aneN4 ligand are deuterated may also suggest the presence of secondary interaction between the –NH groups of [12]aneN4 and [NiIII(O2)]+ moieties, although, such interactions are not obvious in the DFT calculated optimized structure at the employed level of theory.

Efficient catalysts for the anodic oxygen evolution reaction (OER) are critical for electrochemical H2 production. Their design requires structural knowledge of their catalytically active sites and state. Here, we track the atomic-scale structural evolution of well-defined CoOx(OH)y compounds into their catalytically active state during electrocatalytic operation through operando and surface-sensitive X-ray spectroscopy and surface voltammetry, supported by theoretical calculations. We find clear voltammetric evidence that electrochemically reducible near-surface Co3+–O sites play an organizing role for high OER activity. These sites invariably emerge independent of initial metal valency and coordination under catalytic OER conditions. Combining experiments and theory reveals the unified chemical structure motif as µ2-OH-bridged Co2+/3+ ion clusters formed on all three-dimensional cross-linked and layered CoOx(OH)y precursors and present in an oxidized form during the OER, as shown by operando X-ray spectroscopy. Together, the spectroscopic and electrochemical fingerprints offer a unified picture of our molecular understanding of the structure of catalytically active metal oxide OER sites. Knowledge of the active sites in catalysts—including the sites that form under working conditions—is vital for future design and development. Here, the authors track the atomic-scale changes in a series of well-defined cobalt-based oxide electrocatalysts, showing that the structurally distinct catalysts develop a similar structural motif as they transform into the catalytically active state.

The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH4+/NH3 binding. Time-resolved detection of O2 formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NH4Cl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O2 formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn(IV)4 (S3) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) Partial inhibition can be explained by anticooperative (mutually exclusive) NH3 binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the ...

The synthesis and characterization of a hexanuclear cobalt complex 1 involving a non-heme ligand system, L1, supported on a Sn6O6 stannoxane core are reported. Complex 1 acts as a unique catalyst for dioxygen reduction, whose selectivity can be changed from a preferential 4e(-)/4H(+) dioxygen-reduction (to water) to a 2e(-)/2H(+) process (to hydrogen peroxide) only by increasing the temperature from -50 (o)C to 25 (o)C. A variety of spectroscopic methods ((119)Sn-NMR, Magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), SQUID, UV-Vis absorption, resonance Raman (rRaman), and X-ray absorption spectroscopy (XAS) ) coupled with advanced theoretical calculations has been applied for the unambiguous assignment of the geometric and electronic structure of 1. The mechanism of the O2-reduction reaction has been clarified based on kinetic studies on the overall catalytic reaction as well as each step in the catalytic cycle and by low-temperature detection of intermediates. The O2-binding to 1 results in the efficient formation of a stable end-on μ-1,2-peroxodicobalt(III) intermediate 2 at -50 (o)C, followed by a proton-coupled electron-transfer (PCET) reduction to complete the O(2)-to-2H2O cata-lytic conversion in an overall 4e(-)/4H(+) step. In contrast, at higher temperatures (> 20 oC) the constraints provided by the stannoxane core, makes 2 unstable against a preferential proton-transfer (PT) step, leading to the generation of H2O2 by a 2e(-)/2H(+) process. The present study provides deep mechanistic insight into the dioxygen reduction process that should serve as useful and broadly applicable principles for future design of more efficient catalysts in fuel cells

The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the β-barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25 mm3crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge National Laboratory.

Ni–Fe oxyhydroxides are the most active known electrocatalysts for the oxygen evolution reaction (OER) in alkaline electrolytes and are therefore of great scientific and technological importance in the context of electrochemical energy conversion. Here we uncover, investigate, and discuss previously unaddressed effects of conductive supports and the electrolyte pH on the Ni–Fe(OOH) catalyst redox behavior and catalytic OER activity, combining in situ UV–vis spectro-electrochemistry, operando electrochemical mass spectrometry (DEMS), and in situ cryo X-ray absorption spectroscopy (XAS). Supports and pH > 13 strongly enhanced the precatalytic voltammetric charge of the Ni–Fe oxyhydroxide redox peak couple, shifted them more cathodically, and caused a 2–3-fold increase in the catalytic OER activity. Analysis of DEMS-based faradaic oxygen efficiency and electrochemical UV–vis traces consistently confirmed our voltammetric observations, evidencing both a more cathodic O2 release and a more cathodic onset of Ni...

In all organisms that employ oxygenic photosynthesis, the membrane-extrinsic PsbO protein is a functionally important component of photosystem II. To study the previously proposed proton antenna function of carboxylate clusters at the protein-water interface, we combined crystallography and simulations of a truncated cyanobacterial (Thermosynechococcus elongatus) PsbO without peripheral loops. We expressed the PsbO β-barrel heterologously and determined crystal structures at resolutions of 1.15-1.5 Å at 100 K at various pH values and at 297 K and pH 6. (1) Approximately half of the 177 surface waters identified at 100 K are resolved at 297 K, suggesting significant occupancy of specific water sites at room temperature, and loss of resolvable occupancy for other sites. (2) Within a loop region specific to cyanobacterial PsbO, three residues and four waters coordinating a calcium ion are well ordered even at 297 K; the ligation differs for manganese. (3) The crystal structures show water-carboxylate clusters that could facilitate fast Grotthus-type proton transfer along the protein surface and/or store protons. (4) Two carboxylate side chains, which are part of a structural motif interrupting two β-strands and connecting PsbO to photosystem II, are within hydrogen bonding distance at pH 6 (100 K). Simulations indicate coupling between protein structure and carboxylate protonation. The crystal structure determined at 100 K and pH 10 indicates broken hydrogen bonding between the carboxylates and local structural change. At pH 6 and 297 K, both conformations were present in the crystal, suggesting conformational dynamics in the functionally relevant pH regime. Taken together, crystallography and molecular dynamics underline a possible mechanism for pH-dependent structural switching.

The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.

Mimicking the oxygen evolution center Making a synthetic analog of plant photosynthesis is a key goal for exploiting solar energy and replacing fossil fuels. Zhang et al. synthesized a manganese-calcium cluster that looks and acts like the oxygen evolution center in photosystem II (see the Perspective by Sun). The mimic structurally resembles the biological complex, with the notable exception of bridging protein ligands and water-binding sites on a dangling Mn atom. Functionally, however, the cluster's metal center readily undergoes four redox transitions, which contribute to splitting water into oxygen. This and other synthetic mimics will pave the way for developing more efficient catalysts for artificial photosynthesis. Science , this issue p. 690 ; see also p. 635

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-μ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.

Bacteria integrate CO2 reduction and acetyl coenzyme-A (CoA) synthesis in the Wood-Ljungdal pathway. The acetyl-CoA synthase (ACS) active site is a [4Fe4S]-[NiNi] complex (A-cluster). The dinickel site structure (with proximal, p, and distal, d, ions) was studied by X-ray absorption spectroscopy in ACS variants comprising all three protein domains or only the C-terminal domain with the A-cluster. Both variants showed two square-planar Ni(II) sites and an OH- bound at Ni(II)p in oxidized enzyme and a H2O at Ni(I)p in reduced enzyme; a Ni(I)p-CO species was induced by CO incubation and a Ni(II)-CH3- species with an additional water ligand by a methyl group donor. These findings render a direct effect of the N-terminal and middle domains on the A-cluster structure unlikely.

Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pK(a) of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.

Oxidation of water molecules in the photosystem II (PSII) protein complex proceeds at the manganese–calcium complex, which is buried deeply in the lumenal part of PSII. Understanding the PSII function requires knowledge of the intricate coupling between the water-oxidation chemistry and the dynamic proton management by the PSII protein matrix. Here we assess the structural basis for long-distance proton transfer in the interior of PSII and for proton management at its surface. Using the recent high-resolution crystal structure of PSII, we investigate prominent hydrogen-bonded networks of the lumenal side of PSII. This analysis leads to the identification of clusters of polar groups and hydrogen-bonded networks consisting of amino acid residues and water molecules. We suggest that long-distance proton transfer and conformational coupling is facilitated by hydrogen-bonded networks that often involve more than one protein subunit. Proton-storing Asp/Glu dyads, such as the D1-E65/D2-E312 dyad connected to a complex water-wire network, may be particularly important for coupling protonation states to the protein conformation. Clusters of carboxylic amino acids could participate in proton management at the lumenal surface of PSII. We propose that rather than having a classical hydrophobic protein interior, the lumenal side of PSII resembles a complex polyelectrolyte with evolutionary optimized hydrogen-bonding networks. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Photosynthetic water oxidation chemistry at the unique manganese-calcium complex of photosystem II (PSII) is of fundamental importance and serves as a paragon in the development of efficient synthetic catalysts. A recent crystal structure of PSII shows the atoms of the water-oxidizing complex; its Mn4CaO5 core resembles inorganic manganese-calcium oxides. Merging of crystallographic and spectroscopic information reverses radiation-induced modifications at the Mn-complex in silico and facilitates discussion of the O-O bond chemistry. Coordinated proton movements are promoted by a water network connecting the Mn4CaO5 core with the oxidant, a tyrosine radical and one possibly mobile chloride ion. A basic reaction-cycle model predicts an alternating proton and electron removal from the catalytic site, which facilitates energetically efficient water oxidation.

The active site of photosynthetic water oxidation by Photosystem II (PSII) is a manganese-calcium cluster (Mn(4)CaO(5)). A postulated catalytic base is assumed to be crucial. CP43-Arg357, which is a candidate for the identity of this base, is a second-sphere ligand of the Mn(4)-Ca cluster and is located near a putative proton exit pathway, which begins with residue D1-D61. Transient absorption spectroscopy and time-resolved O(2) polarography reveal that in the D1-D61N mutant, the transfer of an electron from the Mn(4)CaO(5) cluster to Y(Z)(OX) and O(2) release during the final step of the catalytic cycle, the S(3)-S(0) transition, proceed simultaneously but are more dramatically decelerated than previously thought (t(1/2) of up to ~50 ms vs a t(1/2) of 1.5 ms in the wild type). Using a bare platinum electrode to record the flash-dependent yields of O(2) from mutant and wild-type PSII has allowed the observation of the kinetics of release of O(2) from extracted thylakoid membranes at various pH values and in the presence of deuterated water. In the mutant, it was possible to resolve a clear lag phase prior to the appearance of O(2), indicating formation of an intermediate before the onset of O(2) formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant, indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regard to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations led to the conclusion that proton expulsion during the initial phase of the S(3)-S(0) transition starts with the deprotonation of the primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O(2) formation chemistry.

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q(B) site in the D1 subunit and thus block the electron transfer from Q(A) to Q(B). Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q(B) site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q(A), which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334-342). This discovery is discussed in the context of proton transfer to the lumen.

Understanding the mechanisms of electron transfer (ET) in photosynthetic reaction centers (RCs) may inspire novel catalysts for sunlight-driven fuel production. The electron exit pathway of type II RCs comprises two quinone molecules working in series and in between a non-heme iron atom with a carboxyl ligand (bicarbonate in photosystem II (PSII), glutamate in bacterial RCs). For decades, the functional role of the iron has remained enigmatic. We tracked the iron site using microsecond-resolution x-ray absorption spectroscopy after laser-flash excitation of PSII. After formation of the reduced primary quinone, Q(A)(-), the x-ray spectral changes revealed a transition (t½ ≈ 150 μs) from a bidentate to a monodentate coordination of the bicarbonate at the Fe(II) (carboxylate shift), which reverted concomitantly with the slower ET to the secondary quinone Q(B). A redox change of the iron during the ET was excluded. Density-functional theory calculations corroborated the carboxylate shift both in PSII and bacterial RCs and disclosed underlying changes in electronic configuration. We propose that the iron-carboxyl complex facilitates the first interquinone ET by optimizing charge distribution and hydrogen bonding within the Q(A)FeQ(B) triad for high yield Q(B) reduction. Formation of a specific priming intermediate by nuclear rearrangements, setting the stage for subsequent ET, may be a common motif in reactions of biological redox cofactors.

Determination of thermodynamic parameters of water oxidation at the photosystem II (PSII) manganese complex is a major challenge. Photothermal beam deflection (PBD) spectroscopy determines enthalpy changes (ΔH) and apparent volume changes which are coupled with electron transfer in the S-state cycle (Krivanek R, Dau H, Haumann M (2008) Biophys J 94: 1890–1903). Recent PBD results on formation of the Q⁻(A)/Y(•+)(Z) radical pair suggest a value of ΔH similar to the free energy change, ΔG, of -540±40 meV previously determined by the analysis of recombination fluorescence, but presently the uncertainty range of ΔH values determined by PBD is still high (±250 meV). In the oxygen-evolving transition, S₃−−S₀, the enthalpy change may be close to zero. A prominent non-thermal signal is associated with both Q⁻(A)/Y(•+)(Z) formation (1 μs) and the S₃−−S₀ transition (~1 ms). The observed (apparent) volume expansion (ΔV of about +40 ų per PSII unit) in the S₃−−S₀ transition seems to revert, at least partially, the contractions on lower S-transitions and may also comprise contributions from O₂ and proton release. The observed volume changes show that the S₃−−S₀ transition is accompanied by significant nuclear movements, which likely are of importance with respect to energetics and mechanism of photosynthetic water oxidation. Detailed PBD studies on all S-transitions will contribute to the progress in PSII research by providing insights not accessible by other spectroscopic methods.

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.

A cobalamin (Cbl) cofactor in corrinoid iron-sulfur protein (CoFeSP) is the primary methyl group donor and acceptor in biological carbon oxide conversion along the reductive acetyl-CoA pathway. Changes of the axial coordination of the cobalt ion within the corrin macrocycle upon redox transitions in aqua-, methyl-, and cyano-Cbl bound to CoFeSP or in solution were studied using X-ray absorption spectroscopy (XAS) at the Co K-edge in combination with density functional theory (DFT) calculations, supported by metal content and cobalt redox level quantification with further spectroscopic methods. Calculation of the highly variable pre-edge X-ray absorption features due to core-to-valence (ctv) electronic transitions, XANES shape analysis, and cobalt-ligand bond lengths determination from EXAFS has yielded models for the molecular and electronic structures of the cobalt sites. This suggested the absence of a ligand at cobalt in CoFeSP in α-position where the dimethylbenzimidazole (dmb) base of the cofactor is bound in Cbl in solution. As main species, (dmb)CoIII(OH2), (dmb)CoII(OH2), and (dmb)CoIII(CH3) sites for solution Cbl and CoIII(OH2), CoII(OH2), and CoIII(CH3) sites in CoFeSP-Cbl were identified. Our data support binding of a serine residue from the reductive-activator protein (RACo) of CoFeSP to the cobalt ion in the CoFeSP-RACo protein complex that stabilizes Co(II). The absence of an α-ligand at cobalt not only tunes the redox potential of the cobalamin cofactor into the physiological range, but is also important for CoFeSP reactivation.

The recent crystallographic structure at 3.0 A resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q(B). The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O(2) evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q(A) and Q(B) were found in the crystallized PSII. We propose that the extra quinones are located in the Q(B) cavity and serve as a PSII intrinsic pool of electron acceptors.

In oxygenic photosynthesis, a complete water oxidation cycle requires absorption of four photons by the chlorophylls of photosystem II (PSII). The photons can be provided successively by applying short flashes of light. Already in 1970, Kok and coworkers [Photochem Photobiol 11:457-475, 1970] developed a basic model to explain the flash-number dependence of O2 formation. The third flash applied to dark-adapted PSII induces the S3--S4--S0 transition, which is coupled to dioxygen formation at a protein-bound Mn4Ca complex. The sequence of events leading to dioxygen formation and the role of Kok's enigmatic S4-state are only incompletely understood. Recently we have shown by time-resolved X-ray spectroscopy that in the S3--S0 transition an interesting intermediate is formed, prior to the onset of O-O bond formation [Haumann et al. Science 310:1019-1021, 2005]. The experimental results of the time-resolved X-ray experiments are discussed. The identity of the reaction intermediate is considered and the question is addressed how the novel intermediate is related to the S4-state proposed in 1970 by Bessel Kok. This leads us to an extension of the classical S-state cycle towards a basic model which describes sequence and interplay of electron and proton abstraction events at the donor side of PSII [Dau and Haumann, Science 312:1471-1472, 2006].

Genetic modification of key residues of photosystems is essential to identify functionally crucial processes by spectroscopic and crystallographic investigation; the required protein stability favours use of thermophilic species. The currently unique thermophilic photosynthetic model organism is the cyanobacterial genus Thermosynechococcus. We report the ability of Thermosynechococcus elongatus to assimilate organic carbon, specifically D-fructose. Growth in the presence of a photosynthesis inhibitor opens the door towards crucial amino acid substitutions in photosystems by the rescue of otherwise lethal mutations. Yet depression of batch-culture growth after 7 days implies that additional developments are needed.

The Mn4Ca complex bound to photosystem II (PSII) is the active site of photosynthetic water oxidation. Its assembly involves binding and light-driven oxidation of manganese, a process denoted as photoactivation. The disassembly of the Mn complex is a thermally activated process involving distinct intermediates. Starting from intermediate states of the disassembly, which was initiated by a temperature jump to 47 degrees C, we photoactivated PSII membrane particles and monitored the activity recovery by O2 polarography and delayed chlorophyll fluorescence measurements. Oxidation state and structural features of the formed intermediates of the Mn complex were assayed by X-ray absorption spectroscopy at the Mn K-edge. The photoactivation time courses, which exhibit a lag phase characteristic of intermediate formation only when starting with the apo-PSII, suggest that within approximately 5 min of photoactivation of apo-PSII, a binuclear Mn complex is formed. It is proposed that a MnIII2(di-mu-oxo) complex is a key intermediate both in the disassembly and in the assembly reaction paths.

Chloride is an important cofactor in photosynthetic water oxidation. It can be replaced by bromide with retention of the oxygen-evolving activity of photosystem II (PSII). Binding of bromide to the Mn(4)Ca complex of PSII in its dark-stable S(1) state was studied by X-ray absorption spectroscopy (XAS) at the Br K-edge in Cl(-)-depleted and Br(-)-substituted PSII membrane particles from spinach. The XAS spectra exclude the presence of metal ions in the first and second coordination spheres of Br(-). EXAFS analysis provided tentative evidence of at least one metal ion, which may be manganese or calcium, at a distance of approximately 5 A to Br(-). The native Cl(-) ion may bind at a similar distance. Accordingly, water oxidation may not require binding of a halide directly to the metal ions of the Mn complex in its S(1) state.

The soluble NAD + -reducing Ni-Fe hydrogenase (SH) from Ralstonia eutropha H16 is remarkable because it cleaves hydrogen in the presence of dioxygen at a unique Ni-Fe active site (Burgdorf et al. (2005) J. Am. Chem. Soc. 127, 576). By X-ray absorption (XAS), FTIR, and EPR spectroscopy, we monitored the structure and oxidation state of its metal centers during H 2 turnover. In NADH-activated protein, a change occurred from the (CN)O 2 Ni II (μ-S)2FeII(CN)3(CO) site dominant in the wild-type SH to a standard-like S 2 Ni II (μ-S)2FeII(CN)2(CO) site as the prevailing species in a specific mutant protein, HoxH-H16L. The wild-type SH primarily was active in H 2 cleavage. The nonstandard reaction mechanism does not involve stable EPR-detectable trivalent Ni oxidation states, namely, the Ni-A,B,C states as observed in standard hydrogenases. In the HoxH-mutant protein H16L, H 2 oxidation was impaired, but H 2 production occurred via a stable Ni-C state (Ni III -H - -Fe II ), suggesting a reaction sequence similar to that of standard hydrogenases. It is proposed that reductive activation by NADH of both wild-type and H16L proteins causes the release of an oxygen species from Ni and is initiated by electron transfer from a [2Fe-2S] cluster in the HoxU subunit that at first becomes reduced by electrons from NADH. Electrons derived from H 2 cleavage, on the other hand, are transferred to NAD + via a different pathway involving a [4Fe-4S] cluster in HoxY, which is reducible only in wild-type SH but not in the H16L variant.

Structural changes upon photoreduction caused by x-ray irradiation of the water-oxidizing tetramanganese complex of photosystem II were investigated by x-ray absorption spectroscopy at the manganese K-edge. Photoreduction was directly proportional to the x-ray dose. It was faster in the higher oxidized S2 state than in S1; seemingly the oxidizing potential of the metal site governs the rate. X-ray irradiation of the S1 state at 15 K initially caused single-electron reduction to S0* accompanied by the conversion of one di-mu-oxo bridge between manganese atoms, previously separated by approximately 2.7 A, to a mono-mu-oxo motif. Thereafter, manganese photoreduction was 100 times slower, and the biphasic increase in its rate between 10 and 300 K with a breakpoint at approximately 200 K suggests that protein dynamics is rate-limiting the radical chemistry. For photoreduction at similar x-ray doses as applied in protein crystallography, halfway to the final Mn(II)4 state the complete loss of inter-manganese distances3 A was observed, even at 10 K, because of the destruction of mu-oxo bridges between manganese ions. These results put into question some structural attributions from recent protein crystallography data on photosystem II. It is proposed to employ controlled x-ray photoreduction in metalloprotein research for: (i) population of distinct reduced states, (ii) estimating the redox potential of buried metal centers, and (iii) research on protein dynamics.

Exposure of Photosystem II (PS II) membrane particles from spinach to a temperature of 47 °C caused the rapid release of the 18 kDa protein in parallel to inactivation of oxygen evolution. Previously, it has been suggested that the first heat-jump response involves rapid Ca release from the Mn complex of O2-evolution, followed by the slower release of (2 + 2) Mn II ions [Pospisil P et al. (2003) Biophys J 84: 1370–1386]. Here, the predicted biphasic Mn II release to the bulk was verified by atomic absorption spectroscopy (AAS). Analysis of laser flash-induced delayed fluorescence transients suggests that the loss of the essential Ca ion from the Mn4Ca complex in the dark is due to the loss of the 18 kDa protein. The S2-state multiline EPR signal of the Mn complex was still generated in heat-treated PS II presumably lacking Ca, but retaining four Mn ions.

Two mechanistic problems of photosynthetic water oxidation at the Mn complex of Photosystem II (PS II) are considered. (I) In the four Mn-oxidizing transitions, any pure Mn oxidation is predicted to cause an increase in redox potential that renders subsequent oxidation steps impossible (redox-potential problem). Formation of unprotonated oxo-bridges may counteract the potential increase. (II) The O–O formation step without any high-pK bases acting as proton acceptors is energetically unfavorable (acceptor-base problem). The pK of oxides in a bridging position between Mn ions may increase drastically upon reduction of Mn in the water-oxidation step (>10 units), thus rendering them favorable proton acceptors. It is proposed that in PS II, in the course of the four oxidizing transitions at least two unprotonated oxo-bridges are formed. Thereby (i) a redox potential increase is prevented and (ii) proton acceptors are prepared for the O–O formation step. Water oxidation in the O–O bond formation step is facilitated by simultaneous Mn reduction and proton transfer to bridging oxides amounting to hydrogen atom or hydride transfer from substrate water to the Mn-oxo core of the Mn complex of PS II.

Structural and electronic changes (oxidation states) of the Mn4Ca complex of photosystem II (PSII) in the water oxidation cycle are of prime interest. For all four transitions between semistable S-states (S0 → S1, S1 → S2, S2 → S3, and S3,4 → S0), oxidation state and structural changes of the Mn complex were investigated by X-ray absorption spectroscopy (XAS) not only at 20 K but also at room temperature (RT) where water oxidation is functional. Three distinct experimental approaches were used: (1) illumination-freeze approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/sampling-XAS method (RT) facilitating particularly direct monitoring of the spectral changes in the S-state cycle. The rate of X-ray photoreduction was quantitatively assessed, and it was thus verified that the Mn ions remained in their initial oxidation state throughout the data collection period (>90%, at 20 K and at RT, for all S-states). Analysis of the complete XANES and EXAFS data sets (20 K and ...

Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.

Methanol:coenzyme M methyltransferase from methanogenic archaea is a cobalamin-dependent enzyme composed of three different subunits: MtaA, MtaB and MtaC. MtaA is a zinc protein that catalyzes the methylation of coenzyme M (HS-CoM) with methylcob(III)alamin. We report zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands. In the presence of coenzyme M, one (N/O)-ligand was replaced by sulfur, most likely due to ligation of the thiol group of coenzyme M. Mutations in His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands. In the His237 → Ala and Cys239 → Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are surprisingly uncritical for coenzyme M binding.

Fingerprints of excitation spectra of chlorophyll (Chl) fluorescence can be used to differentiate 'spectral groups' of microalgae in vivo and in situ in, for example, vertical profiles within a few seconds. The investigated spectral groups of algae (green group, Chlorophyta; blue, Cyanobacteria; brown, Heterokontophyta, Haptophyta, Dinophyta; mixed, Cryptophyta) are each characterised by a specific composition of photosynthetic antenna pigments and, consequently, by a specific excitation spectrum of the Chl fluorescence. Particularly relevant are Chl a, Chl c, phycocyanobilin, phycoerythrobilin, fucoxanthin and peridinin. A laboratory-based instrument and a submersible instrument were constructed containing light-emitting diodes to excite Chl fluorescence in five distinct wavelength ranges. Norm spectra were determined for the four spectral algal groups (several species per group). Using these norm spectra and the actual five-point excitation spectrum of a water sample, a separate estimate of the respective Chl concentration is rapidly obtained for each algal group. The results of dilution experiments are presented. In vivo and in situ measurements are compared with results obtained by HPLC analysis. Depth profiles of the distribution of spectral algal groups taken over a time period of few seconds are shown. The method for algae differentiation described here opens up new research areas, monitoring and supervision tasks related to photosynthetic primary production in aquatic environments.

A binuclear manganese molecular complex [(OH2)(terpy)Mn(μ-O)2Mn(terpy)(OH2)](3+) (1) is the most prominent structural and functional model of the water-oxidizing Mn complex operating in plants and cyanobacteria. Supported on montmorillonite clay and using Ce(IV) as a chemical oxidant, 1 has been reported to be one of the best Mn-based molecular catalysts toward water oxidation. By X-ray absorption spectroscopy and kinetic analysis of the oxygen evolution reaction, we show that [(OH2)(terpy)Mn(μ-O)2Mn(terpy)(OH2)](3+) is transformed into layered type Mn-oxide particles which are the actual water oxidation catalyst.

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.

Detailed information on room-temperature structure and oxidation state of the Photosystem II (PS II) manganese complex is needed to put mechanistic considerations on solid grounds. Because previously this information had not been available, the tetranuclear manganese complex was investigated by X-ray absorption spectroscopy (XAS) on PS II membrane particles at 290 K. Due to methodical progress (collection of XAS spectra within 10 s or less), significant X-ray radiation damage can be avoided; room-temperature XAS investigations on the PS II in its native membrane environment become feasible. Thus, the ambiguity with respect to the mechanistic relevance of low-temperature XAS results is avoidable. At 290 K as well as at 18 K, the manganese complex in its dark-stable state (S(1)-state) seemingly is a Mn(III)(2)Mn(IV)(2) complex comprising two di-mu(2)-oxo bridged binuclear manganese units characterized by the same Mn-Mn distance of 2.71-2.72 A at both temperatures. Most likely, manganese oxidation states and the protonation state of the bridging oxides are fully temperature independent. Remarkably, at room-temperature manganese-ligand distances of 3.10 and 3.65 A are clearly discernible in the EXAFS spectra. The type of bridging assumed to result in Mn-Mn or Mn-Ca distances around 3.1 A is, possibly, temperature-dependent as suggested by distance lengthening upon cooling by 0.13 A. However, mechanistic proposals on photosynthetic water oxidation, which involve the dimer-of-dimers model [Yachandra, V. K., et al. (1993) Science 260, 675-679] are not invalidated by the presented results.

The rise of the chlorophyll fluorescence yield of Photosystem II (PS II) membranes as induced by high-intensity actinic light comprises only two distinct phases: (1) the initial O-J increase and (2) the subsequent J-P increase. Partial inhibition of the PS II donor side by heating or washing procedures which remove peripheral PS II proteins or cofactors of the oxygen-evolving complex results in decrease of magnitude and rate of the J-P phase. The rate constant of the J-P increase is directly proportional to the steady-state rate of oxygen evolution; complete suppression of the J-P phase corresponds to full inhibition. A characteristic dip after J-level is observed only in Tris-washed or severely heated PS II membranes; manganese release correlates with appearance of the dip after J-level as verified by EPR spectroscopy. Presence of stabilizing cosolutes (glycine betaine, sucrose) or addition of donor-side cofactors (bicarbonate, chloride, calcium) to PS II membranes before heating (47 °C, 5 min) diminishes J-P phase suppression and prevents dip appearance, whereas the addition after heating is without effect. In conclusion, analysis of chlorophyll fluorescence transients of PS II membranes is a potentially useful tool for investigations on photosynthetic oxygen evolution. A decreased rate of the J-P phase can be employed as a convenient indicator for partial inhibition of oxygen-evolution activity; the appearance of a dip after J-level is suggestive of manganese release.

Changes in the photosynthetic apparatus occurring during the synchronous cell cycle of the green alga Scenedesmus obliquus are compared to the adaptational response induced by light intensity variations. To investigate and compare these two phenomena, we analyze the polyphasic rise of the chlorophyll fluorescence yield exhibited by plants and cyanobacteria when exposed to high intensity actinic light. Four fluorescence parameters are calculated which are closely related to Photosystem II (PS II) structure and function: ABS/RC, the antenna size of PS II; ϕPO, the quantum yield for reduction of the primary PS II quinone acceptor; qPQ, related to the size of the plastoquinone pool; qEmax, the capacity for pH dependent non-photochemical quenching. The capacity for non-photochemical quenching changes in response to light intensity variations, but it is not affected by the developmental changes occurring during the cell cycle. In contras t, for ABS/RC, ϕPO and qPQ, we observe light induced as well as cell cycle dependent variations. We discuss the relations of the four fluorescence parameters to the molecular organization of the photosynthetic apparatus and its cell cycle and light dependent changes.

By application of microsecond light flashes the oxygen-evolving complex (OEC) was driven through its functional cycle, the S-state cycle. The S-state population distribution obtained by the application of n flashes (n = 0. 6) was determined by analysis of EPR spectra; Mn K-edge X-ray absorption spectra were collected. Taking into consideration the likely statistical error in the data and the variability stemming from the use of three different approaches for the determination of edge positions, we obtained an upshift of the edge position by 0.8-1.5, 0.5-0.9, and 0.6-1.3 eV for the S0-S1, S1-S2, and S2-S3 transitions, respectively, and a downshift by 2.3-3.1 eV for the S3-S0 transition. These results are highly suggestive of Mn oxidation state changes for all four S-state transitions. In the S0-state spectrum, a clearly resolved shoulder in the X-ray spectrum around 6555 eV points toward the presence of Mn(II). We propose that photosynthetic oxygen evolution involves cycling of the photosystem II manganese complex through four distinct oxidation states of this tetranuclear complex: Mn(II)-Mn(III)-Mn(IV)2 in the S0-state, Mn(III)2-Mn(IV)2 in the S1-state, Mn(III)1-Mn(IV)3 in the S2-state, and Mn(IV)4 in the S3-state.

X-ray absorption spectroscopy at the Mn K-edge has been performed on multilayers of photosystem II-enriched fragments of the native thylakoid membrane prepared from a higher plant (spinach) and a unicellular green alga (Scenedesmus obliquus). Spectra collected for various angles between the prevailing orientation of the thylakoid membrane normal and the X-ray electric field vector contain information on the atomic structure of the tetranuclear manganese complex of photosystem II (PS II) and its orientation with respect to the membrane normal. The previously used approach for evaluation of the dichroism of extended X-ray absorption fine structure (EXAFS) spectra [George, G. N., et al. (1989) Science 243, 789-791] is modified, and the following results are obtained for PS II in its dark-stable state (S1-state): (1) structure and orientation of the PS II manganese complexes of green algae and higher plants are highly similiar or fully identical; (2) two 2.7-A vectors, which, most likely, connect the Mn nuclei of a planar Mn2(mu-O2) structure, are at an average angle of 80 degrees +/- 10 degrees with respect to the thylakoid normal; (3) the plane of the Mn2(mu-O2) structures is rather in parallel with the thylakoid plane than perpendicular. Structural models for the oxygen-evolving manganese complex and its orientation in the thylakoid membrane are discussed within the context of the presented results.

The origin of the long-wavelength chlorophyll (Chl) absorption (λpeak > 680 nm) and fluorescence emission (λpeak > 685 nm) has been investigated on Scenedesmus mutants (C-2A′-series, lacking the ability to synthesize chlorophyll in the dark) grown at 0.3 (LL), 10 (ML) and 240 µE s−1 m−2(HL). LL cells are arrested in an early greening state; consequently, ‘Chl availability’ determines the phenotype. LL thylakoids are totally lacking long-wavelength Chl; nonetheless, PS I and PS II are fully functional. Gel electrophoresis and Western blots indicate that four out of seven resolved LHC polypeptides seem to require a high Chl availability for assembly of functional chlorophyll-protein complexes. The PS I core-complex of ML and HL thylakoids contains long-wavelength chlorophylls, but in the PS I core-complex of LL thylakoids these pigments are lacking. We conclude that long-wavelength pigments are only present in the PS I core in the case of high Chl availability. The following hypothesis is discussed: Chl availability determines not only the LHC polypeptide pattern, but also the number of bound Chl molecules per individual pigment-protein complex. Chl-binding at non-obligatory, peripheral sites of the pigment-protein complex results in long-wavelength Chl. In the case of low Chl availability, these sites are not occupied and, therefore, the long-wavelength Chl is absent.

Stable assembly of the major LHC-II proteins (22, 26 kDa) is inhibited in the α-carotenoid-free mutant C-2A′-34 of the green alga Scenedesmus obliquus, whereas other LHC-II proteins (22, 23, 29 kDa) seem to be not affected. We conclude that lutein is essential for assembly of the major LHC-II proteins of S. obliquus, but not for assembly of the minor LHC-II proteins. It is proposed that in the minor LHC-II proteins lutein is replaced by violaxanthin, zeaxanthin and antheraxanthin.