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Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.

Methanotrophic bacteria utilize the nonheme diiron enzyme soluble methane monooxygenase (sMMO) to convert methane to methanol in the first step of their metabolic cycle under copper-limiting conditions. The structure of the sMMO Fe(IV)2 intermediate Q responsible for activating the inert C-H bond of methane (BDE = 104 kcal/mol) remains controversial, with recent studies suggesting both "open" and "closed" core geometries for its active site. In this study, we employ nuclear resonance vibrational spectroscopy (NRVS) to probe the geometric and electronic structure of intermediate Q at cryogenic temperatures. These data demonstrate that Q decays rapidly during the NRVS experiment. Combining data from several years of measurements, we derive the NRVS vibrational features of intermediate Q as well as its cryoreduced decay product. A library of 90 open and closed core models of intermediate Q is generated using density functional theory to analyze the NRVS data of Q and its cryoreduced product as well as prior spectroscopic data on Q. Our analysis reveals that a subset of closed core models reproduce these newly acquired NRVS data as well as prior data. The reaction coordinate with methane is also evaluated using both closed and open core models of Q. These studies show that the potent reactivity of Q toward methane resides in the "spectator oxo" of its Fe(IV)2O2 core, in contrast to nonheme mononuclear Fe(IV)═O enzyme intermediates that H atoms abstract from weaker C-H bonds.

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate- flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free-electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. We show that the flavin in the hydroquinone state is under steric strain in the R2b-NrdI protein complex, presumably tuning its redox potential to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2-interacton site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b- NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

The α-ketoglutarate (αKG)-dependent oxygenases catalyze a diverse range of chemical reactions using a common high-spin FeIV═O intermediate that, in most reactions, abstract a hydrogen atom from the substrate. Previously, the FeIV═O intermediate in the αKG-dependent halogenase SyrB2 was characterized by nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations, which demonstrated that it has a trigonal-pyramidal geometry with the scissile C-H bond of the substrate calculated to be perpendicular to the Fe-O bond. Here, we have used NRVS and DFT calculations to show that the FeIV═O complex in taurine dioxygenase (TauD), the αKG-dependent hydroxylase in which this intermediate was first characterized, also has a trigonal bipyramidal geometry but with an aspartate residue replacing the equatorial halide of the SyrB2 intermediate. Computational analysis of hydrogen atom abstraction by square pyramidal, trigonal bipyramidal, and six-coordinate FeIV═O complexes in two different substrate orientations (one more along [σ channel] and another more perpendicular [π channel] to the Fe-O bond) reveals similar activation barriers. Thus, both substrate approaches to all three geometries are competent in hydrogen atom abstraction. The equivalence in reactivity between the two substrate orientations arises from compensation of the promotion energy (electronic excitation within the d manifold) required to access the π channel by the significantly larger oxyl character present in the pπ orbital oriented toward the substrate, which leads to an earlier transition state along the C-H coordinate.

Significance A new bridging oxygen ligand is incorporated between one of the Mn atoms and Ca in the Mn4Ca cluster during the transition from the one-photon induced S2 intermediate state to the two-photon induced S3 state in the catalytic water oxidation reaction in photosystem II. However, the sequence of events leading to this change is not known. Here we report an X-ray crystallography and spectroscopy study at room temperature using an X-ray free electron laser to collect a “molecular movie” of the structural and oxidation state change steps leading to the insertion of this new oxygen bridge, in the 50 µs to 200 ms time scales after photon absorption, which triggers the S2 → S3 state transition., In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Complementary XFEL methods reveal O2 reaction intermediates and global protein dynamics during Isopenicillin N synthase catalysis., Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Significance A major hurdle in structurally defining the sequence of events that underpin the photointerconversion of phytochromes between their dark-adapted and photoactivated states has been the lack of crystals that undergo these transitions. Here, we describe a crystalline form of the GAF domain from Thermosynechococcus elongatus PixJ within the cyanobacteriochrome subfamily that undergoes reversible photointerconversion and thermal reversion back to the dark-adapted state. Preliminary cryocrystallography of irradiated crystals detected movements of the phycoviolobilin chromophore indicative of a D pyrrole ring rotation. However, X-ray hypersensitivity of both absorbing states might complicate interpretation. Fortunately, we found that PixJ is amenable to serial femtosecond X-ray diffraction methods, which we used to generate a 1.55-Å-resolution model of the dark-adapted state at room temperature., A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs − Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.

Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons., The oxygen-evolving complex in Photosystem II (PSII) catalyzes the light-driven oxidation of water to oxygen and it is still under debate how the water reaches the active site. Here, the authors analyse time-resolved XFEL-based crystal structures of PSII that were determined at room temperature and report the structures of the waters in the putative channels surrounding the active site at various time-points during the reaction cycle and conclude that the O1 channel is the likely water intake pathway and the Cl1 channel the likely proton release pathway.

The α-ketoglutarate (αKG)-dependent oxygenases catalyze a diverse range of chemical reactions using a common high-spin Fe(IV)=O intermediate and, in most reactions, abstracts a hydrogen atom from the substrate. Previously, the Fe(IV)=O intermediate in the αKG-dependent halogenase SyrB2 was characterized by nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations, which demonstrated that it has a trigonal-pyramidal geometry with the scissile C-H bond of the substrate calculated to be perpendicular to the Fe-O bond. Here, we have used NRVS and DFT calculations to show that the Fe(IV)=O complex in taurine dioxygenase (TauD), the αKG-dependent hydroxylase in which this intermediate was first characterized, also has a trigonal bipyramidal geometry, but with an aspartate residue replacing the equatorial halide of the SyrB2 intermediate. Computational analysis of hydrogen atom abstraction by square pyramidal, trigonal bipyramidal, and six-coordinate Fe(IV)=O complexes in two different substrate orientations (one more along [σ channel] and another more perpendicular [π channel] to the Fe-O bond) reveals similar activation barriers. Thus, both substrate approaches to all three geometries are competent in hydrogen atom abstraction. The equivalence in reactivity between the two substrate orientations arises from compensation of the promotion energy (electronic excitation within the d manifold) required to access the π channel by the significantly larger oxyl character present in the pπ orbital oriented towards the substrate, which leads to an earlier transition state along the C-H coordinate.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O(2) activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the [35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S; 112; E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic Fe; II; complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S; 112; E. Two aqua ligands complete the primary coordination sphere of the Fe; II; center with both involved in hydrogen bond networks within Sav. The corresponding Fe; III; protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The Fe; III; protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.

While the synthesis and characterization of {FeNO}7,8,9 complexes have been well documented in heme and nonheme iron models, {FeNO}6 complexes have been less clearly understood. Herein, we report the synthesis and structural and spectroscopic characterization of mononuclear nonheme {FeNO}6 and iron(iii)-nitrito complexes bearing a tetraamido macrocyclic ligand (TAML), such as [(TAML)FeIII(NO)]- and [(TAML)FeIII(NO2)]2-, respectively. First, direct addition of NO(g) to [FeIII(TAML)]- results in the formation of [(TAML)FeIII(NO)]-, which is sensitive to moisture and air. The spectroscopic data of [(TAML)FeIII(NO)]-, such as 1H nuclear magnetic resonance and X-ray absorption spectroscopies, combined with computational study suggest the neutral nature of nitric oxide with a diamagnetic Fe center (S = 0). We also provide alternative pathways for the generation of [(TAML)FeIII(NO)]-, such as the iron-nitrite reduction triggered by protonation in the presence of ferrocene, which acts as an electron donor, and the photochemical iron-nitrite reduction. To the best of our knowledge, the present study reports the first photochemical nitrite reduction in nonheme iron models.

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods that provide significant insight into the correlation of structure with function have now been developed. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin FeIII–OOH non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin FeIV═O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Finally, for several subclasses of non-heme Fe enzymes, binding of the substrate to the FeII site leads to the one-electron reductive activation of O...

FeIII-(hydro)peroxy intermediates have been isolated in two classes of mononuclear non-heme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well defined, due to a lack of vibrational structural data. Nuclear resonance vibrational spectroscopy (NRVS) is an important technique for obtaining vibrational information on these and other intermediates, as it is sensitive to all normal modes with Fe displacement. Here, we present the NRVS spectra of side-on FeIII-peroxy and end-on FeIII-hydroperoxy model complexes and assign these spectra using calibrated DFT calculations. We then use DFT calculations to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe-O-O angle. This study identifies four spectroscopic handles that will enable definition of the binding mode and protonation state of FeIII-peroxy intermediates in mononuclear non-heme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity.

X-ray structures of nonheme {FeNO}6 and iron(iii)-nitrito complexes bearing a tetraamido macrocyclic ligand are reported along with three different generation pathways., While the synthesis and characterization of {FeNO}7,8,9 complexes have been well documented in heme and nonheme iron models, {FeNO}6 complexes have been less clearly understood. Herein, we report the synthesis and structural and spectroscopic characterization of mononuclear nonheme {FeNO}6 and iron(iii)–nitrito complexes bearing a tetraamido macrocyclic ligand (TAML), such as [(TAML)FeIII(NO)]– and [(TAML)FeIII(NO2)]2–, respectively. First, direct addition of NO(g) to [FeIII(TAML)]– results in the formation of [(TAML)FeIII(NO)]–, which is sensitive to moisture and air. The spectroscopic data of [(TAML)FeIII(NO)]–, such as 1H nuclear magnetic resonance and X-ray absorption spectroscopies, combined with computational study suggest the neutral nature of nitric oxide with a diamagnetic Fe center (S = 0). We also provide alternative pathways for the generation of [(TAML)FeIII(NO)]–, such as the iron–nitrite reduction triggered by protonation in the presence of ferrocene, which acts as an electron donor, and the photochemical iron–nitrite reduction. To the best of our knowledge, the present study reports the first photochemical nitrite reduction in nonheme iron models.

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin FeIII–(hydro)-peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native FeII/O2 reaction is evaluated. For the native FeII/O2 reaction, an FeIII–superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of FeIII–superoxo and FeIII–hydroperoxo species in nonheme Fe biochemistry.

Mononuclear nonheme iron(V)-oxo complexes have been reported previously. Herein, we report the first example of a mononuclear nonheme iron(V)-imido complex bearing a tetraamido macrocyclic ligand (TAML), [(TAML)FeV(NTs)]− (1). The spectroscopic characterization of 1 revealed an S = 1/2 Fe(V) oxidation state, an Fe—N bond length of 1.65(4) A, and an Fe—N vibration at 817 cm–1. The reactivity of 1 was demonstrated in C—H bond functionalization and nitrene transfer reactions.

Photosystem II (PSII) catalyzes the light driven oxidation of water into dioxygen, protons and electrons. This reaction takes place at the oxygen evolving complex (OEC) a Mn4CaO5 cluster, through f ...

The synthesis and spectroscopic characterization of [Fe(NO)(N3PyS)]BF4 (3) is presented, the first structural and electronic model of NO-bound cysteine dioxygenase (CDO). The nearly isostructural all-N-donor analog [Fe(NO)(N4Py)](BF4)2 (4) was also prepared, and comparisons of 3 and 4 provide insight regarding the influence of S versus N ligation in {FeNO}7 species. One key difference occurs upon photoirradiation, which causes the fully reversible release of NO from 3, but not from 4.

The reactivity of mononuclear metal-hydroperoxo adducts has fascinated researchers in many areas due to their diverse biological and catalytic processes. In this study, a mononuclear cobalt(III)-peroxo complex bearing a tetradentate macrocyclic ligand, [CoIII(Me3-TPADP)(O2)]+ (Me3-TPADP = 3,6,9-trimethyl-3,6,9-triaza-1(2,6)-pyridinacyclodecaphane), was prepared by reacting [CoII(Me3-TPADP)(CH3CN)2]2+ with H2O2 in the presence of triethylamine. Upon protonation, the cobalt(III)-peroxo intermediate was converted into a cobalt(III)-hydroperoxo complex, [CoIII(Me3-TPADP)(O2H)(CH3CN)]2+. The mononuclear cobalt(III)-peroxo and -hydroperoxo intermediates were characterized by a variety of physicochemical methods. Results of electrospray ionization mass spectrometry clearly show the transformation of the intermediates: the peak at m/z 339.2 assignable to the cobalt(III)-peroxo species disappears with concomitant growth of the peak at m/z 190.7 corresponding to the cobalt(III)-hydroperoxo complex (with bound CH3CN). Isotope labeling experiments further support the existence of the cobalt(III)-peroxo and -hydroperoxo complexes. In particular, the O-O bond stretching frequency of the cobalt(III)-hydroperoxo complex was determined to be 851 cm−1 for 16O2H samples (803 cm−1 for 18O2H samples) and its Co-O vibrational energy was observed at 571 cm−1 for 16O2H samples (551 cm−1 for 18O2H samples; 568 cm−1 for 16O22H samples) by resonance Raman spectroscopy. Reactivity studies performed with the cobalt(III)-peroxo and -hydroperoxo complexes in organic functionalizations reveal that the latter is capable of conducting oxygen atom transfer with an electrophilic character, whereas the former exhibits no oxygen atom transfer reactivity under the same reaction conditions. Alternatively, the cobalt(III)-hydroperoxo complex does not perform hydrogen atom transfer reactions, while analogous low-spin Fe(III)-hydroperoxo complexes are capable of this reactivity. Density function theory calculations indicate that this lack of reactivity is due to the high free energy cost of O-O bond homolysis that would be required to produce the hypothetical Co(IV)-oxo product.

Mononuclear non-haem iron(III)-superoxo species (Fe(III)-O2(-·)) have been implicated as key intermediates in the catalytic cycles of dioxygen activation by non-haem iron enzymes. Although non-haem iron(III)-superoxo species have been trapped and characterized spectroscopically in enzymatic and biomimetic reactions, no structural information has yet been obtained. Here we report the isolation, spectroscopic characterization and crystal structure of a mononuclear side-on (η(2)) iron(III)-superoxo complex with a tetraamido macrocyclic ligand. The non-haem iron(III)-superoxo species undergoes both electrophilic and nucleophilic oxidation reactions, as well as O2-transfer between metal complexes. In the O2-transfer reaction, the iron(III)-superoxo complex transfers the bound O2 unit to a manganese(III) analogue, resulting in the formation of a manganese(IV)-peroxo complex, which is characterized structurally and spectroscopically as a mononuclear side-on (η(2)) manganese(IV)-peroxo complex. The difference in the redox distribution between the metal ions and O2 in iron(III)-superoxo and manganese(IV)-peroxo complexes is rationalized using density functional theory calculations.