Environmental Tuning of Homologs of the Orange Carotenoid Protein-Encoding Gene in the Cyanobacterium Fremyella diplosiphon.

The orange carotenoid protein (OCP) family of proteins are light-activated proteins that function in dissipating excess energy absorbed by accessory light-harvesting complexes, i.e., phycobilisomes (PBSs), in cyanobacteria. Some cyanobacteria contain multiple homologs of the OCP-encoding gene (ocp). Fremyella diplosiphon, a cyanobacterium studied for light-dependent regulation of PBSs during complementary chromatic acclimation (CCA), contains several OCP homologs - two full-length OCPs, three Helical Carotenoid Proteins (HCPs) with homology to the N-terminus of OCP, and one C-terminal domain-like carotenoid protein (CCP) with homology to the C-terminus of OCP. We examined whether these homologs are distinctly regulated in response to different environmental factors, which could indicate distinct functions. We observed distinct patterns of expression for some OCP, HCP, and CCP encoding genes, and have evidence that light-dependent aspects of ocp homolog expression are regulated by photoreceptor RcaE which controls CCA. RcaE-dependent transcriptional regulator RcaC is also involved in the photoregulation of some hcp genes. Apart from light, additional environmental factors associated with cellular redox regulation impact the mRNA levels of ocp homologs, including salt, cold, and disruption of electron transport. Analyses of conserved sequences in the promoters of ocp homologs were conducted to gain additional insight into regulation of these genes. Several conserved regulatory elements were found across multiple ocp homolog promoters that potentially control differential transcriptional regulation in response to a range of environmental cues. The impact of distinct environmental cues on differential accumulation of ocp homolog transcripts indicates potential functional diversification of this gene family in cyanobacteria. These genes likely enable dynamic cellular protection in response to diverse environmental stress conditions in F. diplosiphon.

Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre.

Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP-RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium Rhodobacter sphaeroides.

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, "blue" form with an S2 (11B u+ ) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, "red" form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S2 (11B u+ ) state to BChl a via the Qx state of the latter, accounting for 60% of the total transfer. The elevated S2 (11B u+ ) â†’ Qx transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S2 (11B u+ ) â†’ S0 (11A g- ) carotenoid emission and Qx absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid-protein interactions, including the inferred hydrogen bonding.

Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

Functional characteristics of spirilloxanthin and keto-bearing Analogues in light-harvesting LH2 complexes from Rhodobacter sphaeroides with a genetically modified carotenoid synthesis pathway.

Light-harvesting 2 (LH2) complexes from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N=13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N=13), 2-ketospirilloxanthin (N=14) and 2,2'-diketospirilloxanthin (N=15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N≥13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N≤11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N≥13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas.

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway.

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated C = C bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.

Participation of glutamate-333 of the D1 polypeptide in the ligation of the Mn4CaO5 cluster in photosystem II.

In the 1.9 Å structural model of photosystem II (PDB: 3ARC), the amino acid residue Glu333 of the D1 polypeptide coordinates to the oxygen-evolving Mn4CaO5 cluster. This residue appears to be highly significant in that it bridges the two Mn ions (Mn(B3) and the "dangling" Mn(A4)) that are also bridged by the oxygen atom O5. This oxygen atom has been proposed to be derived from one of two substrate water molecules and to become incorporated into the product dioxygen molecule during the final step in the catalytic cycle. In addition, the backbone nitrogen of D1-Glu333 interacts directly with a nearby Cl⁻ atom. To further explore the influence of this structurally unique residue on the properties of the Mn4CaO5 cluster, the D1-E333Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, and FTIR difference spectroscopy. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild-type. In addition, the oxygen flash yields exhibited normal period-four oscillations having normal S state parameters, although the yields were lower, indicative of the mutant's lower steady-state dioxygen evolution rate of approximately 30% compared to that of the wild-type. The S1 state Mn-XANES and Mn-EXAFS and S2 state multiline EPR signals of purified D1-E333Q PSII core complexes closely resembled those of wild-type, aside from having lower amplitudes. The S(n+1)-minus-S(n) FTIR difference spectra showed only minor alterations to the carbonyl, amide, and carboxylate stretching regions. However, the mutation eliminated a negative peak at 3663 cm⁻¹ in the weakly H-bonding O-H stretching region of the S2-minus-S1 FTIR difference spectrum and caused an approximately 9 cm⁻¹ downshift of the negative feature in this region of the S1-minus-S0 FTIR difference spectrum. We conclude that fully functional Mn4CaO5 clusters assemble in the presence of the D1-E333Q mutation but that the mutation decreases the yield of assembled clusters and alters the H-bonding properties of one or more water molecules or hydroxide groups that are located on or near the Mn4CaO5 cluster and that either deprotonate or form stronger hydrogen bonds during the S0 to S1 and S1 to S2 transitions.

Perturbing the water cavity surrounding the manganese cluster by mutating the residue D1-valine 185 has a strong effect on the water oxidation mechanism of photosystem II.

The active site of water oxidation in Photosystem II (PSII) is a Mn4CaO5 cluster that is located in a cavity between the D1 and CP43 protein subunits by which it is coordinated. The remainder of this cavity is filled with water molecules, which serve as a source of substrate and participate in poorly understood hydrogen bond networks that may modulate the function of the Mn4CaO5 cluster. These water molecules interact with the first and second sphere amino acid ligands to the Mn4CaO5 cluster and some water interacts directly with the Mn4CaO5 cluster. Here, the results of mutations to the amino acids that line the walls of several predicted cavities in the immediate vicinity of the Mn4CaO5 cluster were examined in Synechocystis sp. PCC 6803. Of these, mutations of Val185 in the D1 subunit resulted in the most interesting functional alterations. The hydrophobic D1-Val185 occupies a location contacting water molecules that are positioned between the redox active tyrosine (YZ) and the putative proton gate residue, D1-Asp61, and at a position opposite the oxo bridge atom, O5, of the cluster. Mutations of the residue D1-Val185 were produced, with the intention that the substitute residue would extend into the water cavity that includes H2O molecules that interact with the Mn4CaO5 cluster, amino acid ligands of the Mn4CaO5 cluster, YZ and the chloride co-factor of PSII. Three of these mutants, D1-Val185Asn, D1-Val185Thr, and D1-Val185Phe, were able to accumulate significant levels of charge separating PSII and were characterized using polarographic and fluorescent techniques. Of the three substitutions, the phenylalanine substitution was the most severe with a complete inability to evolve oxygen, despite being able to accumulate PSII and to undergo stable charge separation. The threonine substitution had no apparent effect on oxygen evolution other than a 40% reduction in the steady state rate of O2 production compared to the case of wild-type Synechocystis , due to a reduced ability to accumulate PSII centers. The asparagine substitution produced the most complex phenotype with respect to O2 evolution. Although still able to evolve oxygen, D1-Val185Asn does so less efficiently than wild-type PSII, with a higher miss factor than that for the wild type. Most significantly, asparagine substitution dramatically retards the rate of O2 release and results in an extension of the kinetic lag phase prior to O2 release that is highly reminiscent of the effects of mutations produced at D1-Asp61. The observed effects of the D1-Val185Phe and D1-Val185Asn mutations may be due to alterations in the environment of nearby chloride co-factor of PSII and/or alterations in the hydrogen bond network, perhaps impeding the movement of water to a binding site on the metal cluster.

Synthesis and photophysical properties of chlorins bearing 0-4 distinct meso-substituents.

The presence of substituents at designated sites about the chlorin macrocycle can alter the spectral properties, a phenomenon that can be probed through synthesis. Prior syntheses have provided access to chlorins bearing distinct aryl substituents (individually or collectively) at the 5, 10, and 15-positions, but not the 20-position. A new Western half (5-phenyl-2,3,4,5-tetrahydro-1,3,3-trimethyldipyrrin) has been employed in condensation with an Eastern half (9-bromodipyrromethane-1-carboxaldehyde) followed by oxidative cyclization to give (5% yield) the zinc(II) 20-phenylchlorin. Condensation of the same Western half and a diaryl-substituted Eastern half provided (11% yield) the zinc(II) 5,10,20-triarylchlorin; demetalation with TFA followed by 15-bromination and Suzuki coupling gave the free base 5,10,15,20-tetraarylchlorin. Altogether, 10 new synthetic chlorins have been prepared. The near-UV (B) absorption band of the free base chlorins shifts bathochromically from 389 to 429 nm and that for the zinc chlorins from 398 to 420 nm as the number of meso-aryl rings is increased stepwise from 0-4. The long-wavelength (Q(y)) absorption band undergoes a bathochromic and hypochromic shift upon increase in number of meso-aryl groups. Regardless of the number and positions of the meso-aryl substituents (including "walking a phenyl group around the ring"), the respective fluorescence quantum yields (0.17 to 0.27) and singlet excited-state lifetimes (9.4 to 13.1 ns) are comparable among the free base chlorins and the same is true for the zinc chelates (0.057 to 0.080; 1.2 to 1.6 ns). Density functional theory calculations show that of the frontier molecular orbitals of the chlorin, the energy of the HOMO-1 is the most affected by meso-aryl substituents, undergoing progressive destabilization as the number of meso-aryl groups is increased. The availability of chlorins with 0-4 distinct meso-aryl substituents provides the individual stepping-stones to bridge the known unsubstituted chlorin and the meso-tetraarylchlorins.

Proton transport facilitating water-oxidation: the role of second sphere ligands surrounding the catalytic metal cluster.

The ability of PSII to extract electrons from water, with molecular oxygen as a by-product, is a remarkable biochemical and evolutionary innovation. From an evolutionary perspective, the invention of PSII approximately 2.7 Ga led to the accelerated accumulation of biomass in the biosphere and the accumulation of oxygen in the atmosphere, a combination that allowed for the evolution of a much more complex and extensive biosphere than would otherwise have been possible. From the biochemical and enzymatic perspective, PSII is remarkable because of the thermodynamic and kinetic obstacles that needed to have been overcome to oxidize water as the ultimate photosynthetic electron donor. This article focuses on how proton release is an integral part of how these kinetic and thermodynamic obstacles have been overcome: the sequential removal of protons from the active site of H2O-oxidation facilitates the multistep oxidation of the substrate water at the Mn4CaOx, the catalytic heart of the H2O-oxidation reaction. As noted previously, the facilitated deprotonation of the Mn4CaOx cluster exerts a redox-leveling function preventing the accumulation of excess positive charge on the cluster, which might otherwise hinder the already energetically difficult oxidation of water. Using recent results, including the characteristics of site-directed mutants, the role of the second sphere of amino acid ligands and the associated network of water molecules surrounding the Mn4CaOx is discussed in relation to proton transport in other systems. In addition to the redox-leveling function, a trapping function is assigned to the proton release step occurring immediately prior to the dioxygen chemistry. This trapping appears to involve a yet-to-be clarified gating mechanism that facilitates to coordinated release of a proton from the neighborhood of the active site thereby insuring that the backward charge-recombination reaction does not out-compete the forward reaction of dioxygen chemistry during this final step of H2O-oxidation.

Spectroscopic and functional characterization of cyanobacterium Synechocystis PCC 6803 mutants on the cytoplasmic-side of cytochrome b559 in photosystem II.

We performed spectroscopic and functional characterization on cyanobacterium Synechocystis PCC6803 with mutations of charged residues of the cytoplasmic side of cytochrome (Cyt) b559 in photosystem II (PSII). All of the mutant cells grew photoautotrophically and assembled stable PSII. However, R7Eα, R17Eα and R17Lß mutant cells grew significantly slower and were more susceptible to photoinhibition than wild-type cells. The adverse effects of the arginine mutations on the activity and the stability of PSII were in the following order (R17Lß>R7Eα>R17Eα and R17Aα). All these arginine mutants exhibited normal period-four oscillation in oxygen yield. Thermoluminescence characteristics indicated a slight decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the R7Eα and R17Lß mutant cells. R7Eα and R17Lß PSII core complexes contained predominantly the low potential form of Cyt b559. EPR results indicated the displacement of one of the two axial ligands to the heme of Cyt b559 in R7Eα and R17Lß mutant reaction centers. Our results demonstrate that the electrostatic interactions between these arginine residues and the heme propionates of Cyt b559 are important to the structure and redox properties of Cyt b559. In addition, the blue light-induced nonphotochemical quenching was significantly attenuated and its recovery was accelerated in the R7Lα and R17Lß mutant cells. Furthermore, ultra performance liquid chromatography-mass spectrometry results showed that the PQ pool was more reduced in the R7Eα and R17Lß mutant cells than wild-type cells in the dark. Our data support a functional role of Cyt b559 in protection of PSII under photoinhibition conditions in vivo.

The D1-D61N mutation in Synechocystis sp. PCC 6803 allows the observation of pH-sensitive intermediates in the formation and release of O2 from photosystem II.

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.

Effects of inactivating psbM and psbT on photodamage and assembly of photosystem II in Synechocystis sp. PCC 6803.

PsbM and PsbT have been assigned to electron densities on both photosystem II (PSII) monomers at the PSII dimer interface in X-ray crystallographic structures from Thermosynechoccocus elongatus and T. vulcanus. Our results show that removal of either or both proteins from Synechocystis sp. PCC 6803 resulted in photoautotrophic strains but the DeltaPsbM:DeltaPsbT mutant did not form stable dimers. A CP43-less PSII monomer accumulated in both single mutants, although absence of PsbT destabilized PSII to a greater extent than removing PsbM. Additionally, DeltaPsbT cells exhibited slowed electron transfer between the plastoquinone electron acceptors, Q(A) and Q(B); however, S-state cycling in both mutants was similar to wild type. Oxygen evolution in these mutants rapidly inactivated following exposure to high light where recovery required protein synthesis and could proceed in the dark in DeltaPsbM cells but required light in DeltaPsbT cells. Interestingly, the extent of recovery of oxygen-evolving activity was greatest in the DeltaPsbM:DeltaPsbT strain. We also found recovery required Psb27 in DeltaPsbT cells although, under our conditions, the DeltaPsb27 strain remained similar to wild type. In contrast, the DeltaPsbM:DeltaPsb27 mutant could not assemble PSII beyond a CP43-minus intermediate. Our results suggest essential roles for Psb27 in biogenesis in the DeltaPsbM strain and for repair from photodamage in cells lacking PsbT.

Mutation of arginine 357 of the CP43 protein of photosystem II severely impairs the catalytic S-state cycle of the H2O oxidation complex.

Basic amino acid side chains situated in active sites may mediate critical proton transfers during an enzymatic catalytic cycle. In the case of photosynthetic water oxidation, a strong base is postulated to facilitate the deprotonation of the active site Mn4-Ca cluster, thereby allowing the otherwise thermodynamically constrained transfer of an electron away from the Mn4-Ca cluster to the oxidized redox active tyrosine radical, YZ*, generated by photosynthetic charge separation. Arginine 357 of the CP43 polypeptide may be located in the second coordination shell of the O2-evolving Mn4-Ca cluster of photosystem II (PSII) according to current structural models. An ostensibly conservative substitution mutation, CP43-357K, was investigated using polarographic and fluorescence techniques in evaluating its potential impact on S-state cycling. Cells containing the CP43-357K mutation lost their capacity for autotrophic growth and exhibited a drastic reduction in O2 evolving activity ( approximately 15% of that of the wild type) despite the fact that mutant cells contained more than 80% of the concentration of charge-separating PSII reaction centers and more than half of these contained photooxidizable Mn. Fluorescence kinetics indicated that acceptor side electron transfer, dominated by the transfer of electrons from QA- to QB, was unaffected, but the fraction of centers containing Mn clusters capable of forming the S2 state was reduced to approximately 40% of that of the wild type. Analysis of O2 yields using a bare platinum electrode indicated a severe defect in the S-state cycling properties of the mutant H2O oxidation complexes. Although O2 evolution was delayed to the third flash during a train of single-turnover saturating flashes, the pattern of O2 emission did not exhibit a discernible periodicity indicating a very high miss factor, which was estimated to be approximately 45% compared to the wild-type value of approximately 10%. On the other hand, the multiflash fluorescence measurements indicate that the yield of formation of the S2 state from S1 is diminished by approximately 20%, although this latter estimate is complicated by the presence of damaged PSII centers. Taken together, the experiments indicate that the high miss factor observed during S-state cycling is likely due to a defect in the higher S-state transitions. These results are discussed in relation to the idea that CP43-R357 may serve as a ligand to bicarbonate or as the catalytic base proposed to mediate proton-coupled electron transfer (PCET) in the higher S states of the catalytic cycle of H2O oxidation.