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Dioxygen (O 2 ) is a potent oxidant used by aerobic organisms for energy transduction and critical biosynthetic processes. Numerous metalloenzymes harness O 2 to mediate C-H bond hydroxylation reactions, but most commonly feature iron or copper ions in their active site cofactors. In contrast, many manganese-activated enzymes─such as glutamine synthetase and isocitrate lyase─perform redox neutral chemical transformations and very few are known to activate O 2 or C-H bonds. Here, we report that the dimanganese-metalated form of the cambialistic monooxygenase SfbO (Mn 2 -SfbO) can efficiently mediate enzymatic C-H bond hydroxylation. The activity of the dimanganese form of SfbO toward substrate hydroxylation is comparable to that of its heterobimetallic Mn/Fe form but exhibits distinct kinetic profiles. Kinetic, spectroscopic, and structural studies invoke a mixed-valent dimanganese cofactor (Mn II Mn III ) in O 2 activation and evidence a stoichiometric role for superoxide in maturing an O 2 -inert Mn II 2 cofactor. Computational studies support a hypothesis wherein superoxide addition to the Mn II 2 cofactor installs a critical bridging hydroxide ligand that stabilizes higher-valent manganese oxidation states. These findings establish the viability of proteinaceous dimanganese cofactors in mediating complex, multistep redox transformations.

The low reduction potentials required for the reduction of dinitrogen (N 2 ) render metal-based nitrogen-fixation catalysts vulnerable to irreversible damage by dioxygen (O 2 ) 1-3 . Such O 2 sensitivity represents a major conundrum for the enzyme nitrogenase, as a large fraction of nitrogen-fixing organisms are either obligate aerobes or closely associated with O 2 -respiring organisms to support the high energy demand of catalytic N 2 reduction 4 . To counter O 2 damage to nitrogenase, diazotrophs use O 2 scavengers, exploit compartmentalization or maintain high respiration rates to minimize intracellular O 2 concentrations 4-8 . A last line of damage control is provided by the 'conformational protection' mechanism 9 , in which a [2Fe:2S] ferredoxin-family protein termed FeSII (ref.  10 ) is activated under O 2 stress to form an O 2 -resistant complex with the nitrogenase component proteins 11,12 . Despite previous insights, the molecular basis for the conformational O 2 protection of nitrogenase and the mechanism of FeSII activation are not understood. Here we report the structural characterization of the Azotobacter vinelandii FeSII-nitrogenase complex by cryo-electron microscopy. Our studies reveal a core complex consisting of two molybdenum-iron proteins (MoFePs), two iron proteins (FePs) and one FeSII homodimer, which polymerize into extended filaments. In this three-protein complex, FeSII mediates an extensive network of interactions with MoFeP and FeP to position their iron-sulphur clusters in catalytically inactive but O 2 -protected states. The architecture of the FeSII-nitrogenase complex is confirmed by solution studies, which further indicate that the activation of FeSII involves an oxidation-induced conformational change., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

The S 3 state is currently the last observable intermediate prior to O-O bond formation at the oxygen-evolving complex (OEC) of Photosystem II, and its electronic structure has been assigned to a homovalent Mn IV 4 core with an S = 3 ground state. While structural interpretations based on the EPR spectroscopic features of the S 3 state provide valuable mechanistic insight, corresponding synthetic and spectroscopic studies on tetranuclear complexes mirroring the Mn oxidation states of the S 3 state remain rare. Herein, we report the synthesis and characterization by XAS and multifrequency EPR spectroscopy of a Mn IV 4 O 4 cuboidal complex as a spectroscopic model of the S 3 state. Results show that this Mn IV 4 O 4 complex has an S = 3 ground state with isotropic 55 Mn hyperfine coupling constants of -75, -88, -91, and 66 MHz. These parameters are consistent with an αααβ spin topology approaching the trimer-monomer magnetic coupling model of pseudo-octahedral Mn IV centers. Importantly, the spin ground state changes from S = 1/2 to S = 3 as the OEC is oxidized from the S 2 state to the S 3 state. This same spin state change is observed following oxidation of the previously reported Mn III Mn IV 3 O 4 cuboidal complex to the Mn IV 4 O 4 complex described here. This sets a synthetic precedent for the observed low-spin to high-spin conversion in the OEC.

High-valent Ru V -oxo intermediates have long been proposed in catalytic oxidation chemistry, but investigations into their electronic and chemical properties have been limited due to their reactive nature and rarity. The incorporation of Ru into the [Co 3 O 4 ] subcluster via the single-step assembly reaction of Co II (OAc) 2 (H 2 O) 4 (OAc = acetate), perruthenate (RuO 4 - ), and pyridine (py) yielded an unprecedented Ru(O)Co 3 (μ 3 -O) 4 (OAc) 4 (py) 3 cubane featuring an isolable, yet reactive, Ru V -oxo moiety. EPR, ENDOR, and DFT studies reveal a valence-localized [Ru V ( S = 1/2)Co III 3 ( S = 0)O 4 ] configuration and non-negligible covalency in the cubane core. Significant oxyl radical character in the Ru V -oxo unit is experimentally demonstrated by radical coupling reactions between the oxo cubane and both 2,4,6-tri- tert -butylphenoxyl and trityl radicals. The oxo cubane oxidizes organic substrates and, notably, reacts with water to form an isolable μ-oxo bis-cubane complex [(py) 3 (OAc) 4 Co 3 (μ 3 -O) 4 Ru]-O-[RuCo 3 (μ 3 -O) 4 (OAc) 4 (py) 3 ]. Redox activity of the Ru V -oxo fragment is easily tuned by the electron-donating ability of the distal pyridyl ligand set at the Co sites demonstrating strong electronic communication throughout the entire cubane cluster. Natural bond orbital calculations reveal cooperative orbital interactions of the [Co 3 O 4 ] unit in supporting the Ru V -oxo moiety via a strong π-electron donation.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O 2 ) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O 2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O 2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, β-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-β-gal activity and DNA damage response, cell cycle arrest in G 2 /M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-β, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.

The bacterial protein complex Mnx contains a multicopper oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO 2 biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu 2+ , the electron acceptor. Indeed the type 1 Cu 2+ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO 2 formation from MnO 2 nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pK a > 8.6 deprotonation, which is assigned to Mn(II)-bound H 2 O; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(μ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pK a 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(μ-OH) 2 Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(μ-O) 2 Mn(IV) or an oligomer, which subsequently nucleates MnO 2 nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper .

Lung diseases, such as bronchopulmonary dysplasia (BPD), wheezing, and asthma, remain significant causes of morbidity and mortality in the pediatric population, particularly in the setting of premature birth. Pulmonary outcomes in these infants are highly influenced by perinatal exposures including prenatal inflammation, postnatal intensive care unit interventions, and environmental agents. Here, there is strong evidence that perinatal supplemental oxygen administration has significant effects on pulmonary development and health. This is of particular importance in the preterm lung, where premature exposure to room air represents a hyperoxic insult that may cause harm to a lung primed to develop in a hypoxic environment. Preterm infants are also subject to increased episodes of hypoxia, which may also result in pulmonary damage and disease. Here, we summarize the current understanding of the effects of oxygen on the developing lung and how low vs. high oxygen may predispose to pulmonary disease that may extend even into adulthood. Better understanding of the underlying mechanisms will help lead to improved care and outcomes in this vulnerable population.

The laboratory synthesis of the oxygen-evolving complex (OEC) of photosystem II has been the objective of synthetic chemists since the early 1970s. However, the absence of structural information on the OEC has hampered these efforts. Crystallographic reports on photosystem II that have been appearing at ever-improving resolution over the past ten years have finally provided invaluable structural information on the OEC and show that it comprises a [Mn(3)CaO(4)] distorted cubane, to which is attached a fourth, external Mn atom, and the whole unit attached to polypeptides primarily by aspartate and glutamate carboxylate groups. Such a heterometallic Mn/Ca cubane with an additional metal attached to it has been unknown in the literature. This paper reports the laboratory synthesis of such an asymmetric cubane-containing compound with a bound external metal atom, [(1)]. All peripheral ligands are carboxylate or carboxylic acid groups. Variable-temperature magnetic susceptibility data have established 1 to possess an S = 9/2 ground state. EPR spectroscopy confirms this, and the Davies electron nuclear double resonance data reveal similar hyperfine couplings to those of other Mn(IV) species, including the OEC S(2) state. Comparison of the X-ray absorption data with those for the OEC reveal 1 to possess structural parameters that make it a close structural model of the asymmetric-cubane OEC unit. This geometric and electronic structural correspondence opens up a new front in the multidisciplinary study of the properties and function of this important biological unit.

We report the generation and characterization of a new high-spin iron(IV)-oxo complex supported by a trigonal nonheme pyrrolide platform. Oxygen-atom transfer to [(tpa(Mes))Fe(II)](-) (tpa(Ar) = tris(5-arylpyrrol-2-ylmethyl)amine) in acetonitrile solution affords the Fe(III)-alkoxide product [(tpa(Mes2MesO))Fe(III)](-) resulting from intramolecular C-H oxidation with no observable ferryl intermediates. In contrast, treatment of the phenyl derivative [(tpa(Ph))Fe(II)](-) with trimethylamine N-oxide in acetonitrile solution produces the iron(IV)-oxo complex [(tpa(Ph))Fe(IV)(O)](-) that has been characterized by a suite of techniques, including mass spectrometry as well as UV-vis, FTIR, Mössbauer, XAS, and parallel-mode EPR spectroscopies. Mass spectral, FTIR, and optical absorption studies provide signatures for the iron-oxo chromophore, and Mössbauer and XAS measurements establish the presence of an Fe(IV) center. Moreover, the Fe(IV)-oxo species gives parallel-mode EPR features indicative of a high-spin, S = 2 system. Preliminary reactivity studies show that the high-spin ferryl tpa(Ph) complex is capable of mediating intermolecular C-H oxidation as well as oxygen-atom transfer chemistry., (© 2012 American Chemical Society)

We have examined the antiferromagneticly coupled bis(μ-oxo)dimanganese(IV) complex [Mn(2)O(2)(salpn)(2)] (1) with (55)Mn solid-state NMR at cryogenic temperatures and first-principle theory. The extracted values of the (55)Mn quadrupole coupling constant, C(Q), and its asymmetry parameter, η(Q), for 1 are 24.7 MHz and 0.43, respectively. Further, there was a large anisotropic contribution to the shielding of each Mn(4+), i.e. a Δσ of 3375 ppm. Utilizing broken symmetry density functional theory, the predicted values of the electric field gradient (EFG) or equivalently the C(Q) and η(Q) at ZORA, PBE QZ4P all electron level of theory are 23.4 MHz and 0.68, respectively, in good agreement with experimental observations.

In the recent X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is assigned as a ligand of the O2-evolving Mn4Ca cluster. In this communication, a preliminary characterization of the CP43-Glu354Gln mutant of the cyanobacterium Synechocystis sp. PCC 6803 is presented. The steady-state rate of O2 evolution in the mutant cells is only approximately 20% compared with the wild-type, but the kinetics of O2 release are essentially unchanged and the O2-flash yields show normal period-four oscillations, albeit with lower overall intensity. Purified PSII particles exhibit an essentially normal S2 state multiline electron paramagnetic resonance (EPR) signal, but exhibit a substantially altered S2-minus-S1 Fourier transform infrared (FTIR) difference spectrum. The intensities of the mutant EPR and FTIR difference spectra (above 75% compared with wild-type) are much greater than the O2 signals and suggest that CP43-Glu354Gln PSII reaction centres are heterogeneous, with a minority fraction able to evolve O2 with normal O2 release kinetics and a majority fraction unable to advance beyond the S2 or S3 states. The S2-minus-S1 FTIR difference spectrum of CP43-Glu354Gln PSII particles is altered in both the symmetric and asymmetric carboxylate stretching regions, implying either that CP43-Glu354 is exquisitely sensitive to the increased charge that develops on the Mn4Ca cluster during the S1-->S2 transition or that the CP43-Glu354Gln mutation changes the distribution of Mn(III) and Mn(IV) oxidation states within the Mn4Ca cluster in the S1 and/or S2 states.

The proximity of the calcium/strontium binding site of the oxygen evolving complex (OEC) of photosystem II (PSII) to the paramagnetic Mn cluster is explored with (87)Sr three-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy. CW-EPR spectra of Sr(2+)-substituted Ca(2+)-depleted PSII membranes show the modified g = 2 multiline EPR signal as previously reported. We performed three-pulse ESEEM on this modified multiline signal of the Mn cluster using natural abundance Sr and (87)Sr, respectively. Three-pulse ESEEM of the natural abundance Sr sample exhibits no detectable modulation by the 7% abundance (87)Sr. On the other hand, that of the (87)Sr enriched (93%) sample clearly reveals modulation arising from the I = (9)/(2) (87)Sr nucleus weakly magnetically coupled to the Mn cluster. Using a simple point dipole approximation for the electron spin, analysis of the (87)Sr ESEEM modulation depth via an analytic expression suggests a Mn-Ca (Sr) distance of 4.5 A. Simulation of three-pulse ESEEM with a numerical matrix diagonalization procedure gave good agreement with this analytical result. A more appropriate tetranuclear magnetic/structural model for the Mn cluster converts the 4.5 A point dipole distance to a 3.8-5.0 A range of distances. DFT calculations of (43)Ca and (87)Sr quadrupolar interactions on Ca (and Sr substituted) binding sites in various proteins suggest that the lack of the nuclear quadrupole induced splitting in the ESEEM spectrum of (87)Sr enriched PSII samples is related to a very high degree of symmetry of the ligands surrounding the Sr(2+) ion in the substituted Ca site. Numerical simulations show that moderate (87)Sr quadrupolar couplings decrease the envelope modulation relative to the zero quadrupole case, and therefore we consider that the 3.8-5.0 A range obtained without quadrupolar coupling included in the simulation represents an upper limit to the actual manganese-calcium distance. This (87)Sr pulsed EPR spectroscopy provides independent direct evidence that the calcium/strontium binding site is close to the Mn cluster in the OEC of PSII.

Previously, using acetate deuterated in the methyl hydrogen positions, we showed that acetate binds in close proximity to the Mn cluster/Y(.)(z) tyrosine dual spin complex in acetate-inhibited photosystem II (PSII) preparations exhibiting the "split" EPR signal arising from the S(2)-Y(.)(z) interaction [Force, D. A.; Randall, D. W.; Britt, R. D. Biochemistry 1997, 36, 12062-12070]. By using paramagnetic NO to quench the paramagnetism of Y(.)(z), we are able to observe the ESEEM spectrum of deuterated acetate interacting with only the Mn cluster. A good fit of the ESEEM data indicates two (2)H dipolar hyperfine couplings of 0.097 MHz and one of 0.190 MHz. Modeling of these dipolar interactions, using our "dangler" 3 + 1 model for the S(2)-state of the Mn cluster, reveals distances consistent with direct ligation of acetate to the Mn cluster. As acetate inhibition is competitive with the essential cofactor Cl(-), this suggests that Cl(-) ligates directly to the Mn cluster. The effect of acetate binding on the structure of the Mn cluster is investigated by comparing the Mn-histidine coupling in NO/acetate-treated PSII and untreated PSII using ESEEM. We find that the addition of acetate and NO does not affect the histidine ligation to the Mn cluster. We also investigate the ability of acetate to access Y(.)(z) in Mn-depleted PSII, a PSII preparation expected to be more solvent accessible than intact PSII. We detect no coupling between Y(.)(z) and acetate. We have previously shown that small alcohols such as methanol can ligate to the Mn cluster with ease, while larger alcohols such as 2-propanol, as well as DMSO, are excluded [Force, D. A.; Randall, D. W.; Lorigan, G. A.; Clemens, K. L.; Britt, R. D. J. Am. Chem. Soc. 1998, 120, 13321-13333]. We probe the effect of acetate binding on the ability of methanol and DMSO to bind to the Mn cluster. We find that methanol is able to bind to the Mn cluster in the presence of acetate. We detect no DMSO binding in the presence of acetate. Thus, acetate binding does not increase the affinity or accessibility for DMSO binding at the Mn cluster. We also explore the possibility that the acetate binding site is also a binding site for substrate water. By comparing the ratioed three-pulse ESEEM spectra of a control, untreated PSII sample in 50% D(2)O to an NO/acetate-treated PSII sample in 50% D(2)O, we find that the binding of acetate to the oxygen evolving complex of photosystem II displaces deuterons bound very closely to the Mn cluster.

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).

Photosystem II uses visible light to drive the oxidation of water, resulting in bioactivated electrons and protons, with the production of molecular oxygen as a byproduct. This water-splitting reaction is carried out by a manganese cluster/tyrosine radial ensemble, the oxygen -evolving complex. Although conventional continuous-wave, perpendicular -polarization electron paramagnetic resonance (EPR) spectroscopy has significantly advanced our knowledge of the structure and function of the oxygen-evolving complex, significant additional information can be obtained with the application of additional EPR methodologies. Specifically, parallel-polarization EPR spectroscopy can be use to obtain highly resolved EPR spectra of integer spin Mn species, and pulsed EPR spectroscopy with electron spin echo-based sequences, such as electron spin echo envelope modulation and electron spin echo-electron nuclear double resonance, can be used to measure weak interactions obscured in continuous-wave spectroscopy by inhomogeneous broadening.

The amplitude of the g = 2 Mn 'multiline' EPR signal of the S2 state of the photosynthetic oxygen-evolving complex varies inversely with temperature, indicating that this signal arises from a ground spin state. Electron spin echo experiments at temperatures of 4.2 K and 1.4 K show such Curie-law behavior of the g = 2 multiline EPR signal, as do continuous-wave EPR experiments performed at a non-saturating microwave power in the range from 15.0 K to 4.2 K.

The g = 4 and g = 2 multiline EPR signals arising from the Mn cluster of the photosynthetic oxygen-evolving complex (OEC) in the S2 state were studied in preparations of oriented photosystem II (PSII) membranes. The ammonia-modified forms of these two signals were also examined. The g = 4 signal obtained in oriented PSII membranes treated with NH4Cl at pH 7.5 displays at least 16 partially resolved Mn hyperfine transitions with a regular spacing of 36 G [Kim, D.H., Britt, R.D., Klein, M.P., & Sauer, K. (1990) J. Am. Chem. Soc. 112, 9389-9391]. The observation of this g = 4 "multiline signal" provides strong spectral evidence for a tetranuclear Mn origin for the g = 4 signal and is strongly suggestive of a model in which different spin state configurations of a single exchange-coupled Mn cluster give rise to the g = 4 and g = 2 multiline signals. A simulation shows the observed spectrum to be consistent with an S = 3/2 or S = 5/2 state of a tetranuclear Mn complex. The resolution of hyperfine structure on the NH3-modified g = 4 signal is strongly dependent on sample orientation, with no resolved hyperfine structure when the membrane normal is oriented perpendicular to the applied magnetic field. The dramatic NH3-induced changes in the g = 4 signal resolved in the spectra of oriented samples are suggestive that NH3 binding at the Cl- site of the OEC may represent direct coordination of NH3 to the Mn cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

A Mn-containing enzyme complex is involved in the oxidation of H2O to O2 in algae and higher plants. X-ray absorption spectroscopy is well suited for studying the structure and function of Mn in this enzyme complex. Results of X-ray K-edge and extended X-ray absorption fine structure (EXAFS) studies of Mn in the S1 and S2 states of the photosynthetic O2-evolving complex in photosystem II preparations from spinach are presented in this paper. The S2 state was prepared by illumination at 190 K or by illumination at 277 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); these are protocols that limit the photosystem II reaction center to one turnover. Both methods produce an S2 state characterized by a multiline electron paramagnetic resonance (EPR) signal. An additional protocol, illumination at 140 K, produces as a state characterized by the g = 4.1 EPR signal. We have previously observed a shift to higher energy in the X-ray absorption K-edge energy of Mn upon advancement from the dark-adapted S1 state to the S2 state produced by illumination at 190 K [Goodin, D. B., Yachandra, V. K., Britt, R. D., Sauer, K., & Klein, M. P. (1984) Biochim. Biophys. Acta 767, 209-216]. The Mn K-edge spectrum of the 277 K illuminated sample is similar to that produced at 190 K, indicating that the S2 state is similar when produced at 190 or 277 K.(ABSTRACT TRUNCATED AT 250 WORDS)

The Mn donor complex in the S1 and S2 states and the iron-quinone acceptor complex (Fe2+-Q) in O2-evolving photosystem II (PS II) preparations from a thermophilic cyanobacterium, Synechococcus sp., have been studied with X-ray absorption spectroscopy and electron paramagnetic resonance (EPR). Illumination of these preparations at 220-240 K results in formation of a multiline EPR signal very similar to that assigned to a Mn S2 species observed in spinach PS II, together with g = 1.8 and 1.9 EPR signals similar to the Fe2+-QA- acceptor signals seen in spinach PS II. Illumination at 110-160 K does not produce the g = 1.8 or 1.9 EPR signals, nor the multiline or g = 4.1 EPR signals associated with the S2 state of PS II in spinach; however, a signal which peaks at g = 1.6 appears. The most probable assignment of this signal is an altered configuration of the Fe2+-QA- complex. In addition, no donor signal was seen upon warming the 140 K illuminated sample to 215 K. Following continuous illumination at temperatures between 140 and 215 K, the average X-ray absorption Mn K-edge inflection energy changes from 6550 eV for a dark-adapted (S1) sample to 6551 eV for the illuminated (S2) sample. The shift in edge inflection energy indicates an oxidation of Mn, and the absolute edge inflection energies indicate an average Mn oxidation state higher than Mn(II). Upon illumination a significant change was observed in the shape of the features associated with 1s to 3d transitions. The S1 spectrum resembles those of Mn(III) complexes, and the S2 spectrum resembles those of Mn(IV) complexes. The extended X-ray absorption fine structure (EXAFS) spectrum of the Mn complex is similar in the S1 and S2 states. Simulations indicate O or N ligands at 1.75 +/- 0.05 A, transition metal neighbor(s) at 2.73 +/- 0.05 A, which are assumed to be Mn, and terminal ligands which are probably N and O at a range of distances around 2.2 A. The Mn-O bond length of 1.75 A and the transition metal at 2.7 A indicate the presence of a di-mu-oxo-bridged Mn structure. Simulations indicate that a symmetric tetranuclear cluster is unlikely to be present, while binuclear, trinuclear, or highly distorted tetranuclear structures are possible. The striking similarity of these results to those from spinach PS II suggests that the structure of the Mn complex is largely conserved across evolutionarily diverse O2-evolving photosynthetic species.