ABSTRACT
In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3â[S4]âS0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2âS3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.
Subject(s)
Oxygen , Photosynthesis , Photosystem II Protein Complex , Oxidation-Reduction , Oxygen/chemistry , Oxygen/metabolism , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Protons , Water/chemistry , Water/metabolism , Manganese/chemistry , Manganese/metabolism , Calcium/chemistry , Calcium/metabolism , Peroxides/metabolismABSTRACT
Hydrogenases display a wide range of catalytic rates and biases in reversible hydrogen gas oxidation catalysis. The interactions of the iron-sulfur-containing catalytic site with the local protein environment are thought to contribute to differences in catalytic reactivity, but this has not been demonstrated. The microbe Clostridium pasteurianum produces three [FeFe]-hydrogenases that differ in "catalytic bias" by exerting a disproportionate rate acceleration in one direction or the other that spans a remarkable 6 orders of magnitude. The combination of high-resolution structural work, biochemical analyses, and computational modeling indicates that protein secondary interactions directly influence the relative stabilization/destabilization of different oxidation states of the active site metal cluster. This selective stabilization or destabilization of oxidation states can preferentially promote hydrogen oxidation or proton reduction and represents a simple yet elegant model by which a protein catalytic site can confer catalytic bias.
Subject(s)
Hydrogen/metabolism , Hydrogenase/metabolism , Catalysis , Clostridium/enzymology , Oxidation-Reduction , X-Ray DiffractionABSTRACT
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 O2 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.
Subject(s)
Oxygenases/chemistry , Temperature , Methylosinus trichosporium/enzymology , Models, Molecular , Oxidation-Reduction , Oxygenases/metabolism , Solubility , X-RaysABSTRACT
Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.
Subject(s)
Azotobacter vinelandii/enzymology , Molybdoferredoxin/chemistry , Nitrogenase/chemistry , Protein Conformation , Protons , Catalysis , Crystallography, X-Ray , Electron Transport , Molybdoferredoxin/metabolism , Nitrogenase/metabolism , Oxidation-ReductionABSTRACT
The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of ß2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.
Subject(s)
Chemistry, Physical/instrumentation , Crystallography, X-Ray/methods , Protein Conformation , Proteins/chemistry , Crystallization , Electrons , Lasers , Models, Molecular , Myoglobin/chemistry , RNA Polymerase II/chemistry , Receptors, Adrenergic, beta-2/chemistry , Reproducibility of Results , Synchrotrons , X-Ray Diffraction/methods , X-RaysABSTRACT
The reduction of substrates catalyzed by nitrogenase normally requires nucleotide-dependent Fe protein delivery of electrons to the MoFe protein, which contains the active site FeMo cofactor. Here, it is reported that independent substitution of three amino acids (ß-98(TyrâHis), α-64(TyrâHis), and ß-99(PheâHis)) located between the P cluster and FeMo cofactor within the MoFe protein endows it with the ability to reduce protons to H2, azide to ammonia, and hydrazine to ammonia without the need for Fe protein or ATP. Instead, electrons can be provided by the low-potential reductant polyaminocarboxylate-ligated Eu(II) (Em values of -1.1 to -0.84 V vs the normal hydrogen electrode). The crystal structure of the ß-98(TyrâHis) variant MoFe protein was determined, revealing only small changes near the amino acid substitution that affect the solvent structure and the immediate vicinity between the P cluster and the FeMo cofactor, with no global conformational changes observed. Computational normal-mode analysis of the nitrogenase complex reveals coupling in the motions of the Fe protein and the region of the MoFe protein with these three amino acids, which suggests a possible mechanism for how Fe protein might communicate subtle changes deep within the MoFe protein that profoundly affect intramolecular electron transfer and substrate reduction.
Subject(s)
Azotobacter vinelandii/enzymology , Bacterial Proteins/chemistry , Coenzymes/chemistry , Computer Simulation , Iron/chemistry , Molybdenum/chemistry , Nitrogenase/chemistry , Adenosine Triphosphate/chemistry , Amino Acid Substitution , Azotobacter vinelandii/genetics , Bacterial Proteins/genetics , Coenzymes/genetics , Mutation, Missense , Nitrogenase/geneticsABSTRACT
Biological magnetic field sensing that gives rise to physiological responses is of considerable importance in quantum biology. The radical pair mechanism (RPM) is a fundamental quantum process that can explain some of the observed biological magnetic effects. In magnetically sensitive radical pair (RP) reactions, coherent spin dynamics between singlet and triplet pairs are modulated by weak magnetic fields. The resulting singlet and triplet reaction products lead to distinct biological signaling channels and cellular outcomes. A prevalent RP in biology is between flavin semiquinone and superoxide (O2 â¢-) in the biological activation of molecular oxygen. This RP can result in a partitioning of reactive oxygen species (ROS) products to form either O2 â¢- or hydrogen peroxide (H2O2). Here, we examine magnetic sensing of recombinant human electron transfer flavoenzyme (ETF) reoxidation by selectively measuring O2 â¢- and H2O2 product distributions. ROS partitioning was observed between two static magnetic fields at 20 nT and 50 µT, with a 13% decrease in H2O2 singlet products and a 10% increase in O2 â¢- triplet products relative to 50 µT. RPM product yields were calculated for a realistic flavin/superoxide RP across the range of static magnetic fields, in agreement with experimental results. For a triplet born RP, the RPM also predicts about three times more O2 â¢- than H2O2, with experimental results exhibiting about four time more O2 â¢- produced by ETF. The method presented here illustrates the potential of a novel magnetic flavoprotein biological sensor that is directly linked to mitochondria bioenergetics and can be used as a target to study cell physiology.
ABSTRACT
Azotobacter vinelandii nitrogenase Fe protein (Av2) provides a rare opportunity to investigate a [4Fe-4S] cluster at three oxidation levels in the same protein environment. Here, we report the structural and vibrational changes of this cluster upon reduction using a combination of NRVS and EXAFS spectroscopies and DFT calculations. Key to this work is the synergy between these three techniques as each generates highly complementary information and their analytical methodologies are interdependent. Importantly, the spectroscopic samples contained no glassing agents. NRVS and DFT reveal a systematic 10-30 cm(-1) decrease in Fe-S stretching frequencies with each added electron. The "oxidized" [4Fe-4S](2+) state spectrum is consistent with and extends previous resonance Raman spectra. For the "reduced" [4Fe-4S](1+) state in Fe protein, and for any "all-ferrous" [4Fe-4S](0) cluster, these NRVS spectra are the first available vibrational data. NRVS simulations also allow estimation of the vibrational disorder for Fe-S and Fe-Fe distances, constraining the EXAFS analysis and allowing structural disorder to be estimated. For oxidized Av2, EXAFS and DFT indicate nearly equal Fe-Fe distances, while addition of one electron decreases the cluster symmetry. However, addition of the second electron to form the all-ferrous state induces significant structural change. EXAFS data recorded to k = 21 Å(-1) indicates a 1:1 ratio of Fe-Fe interactions at 2.56 Å and 2.75 Å, a result consistent with DFT. Broken symmetry (BS) DFT rationalizes the interplay between redox state and the Fe-S and Fe-Fe distances as predominantly spin-dependent behavior inherent to the [4Fe-4S] cluster and perturbed by the Av2 protein environment.
Subject(s)
Oxidoreductases/chemistry , Quantum Theory , Fourier Analysis , Models, Molecular , Oxidation-Reduction , VibrationABSTRACT
Upside-down jellyfish (Cassiopea sp.) are mostly sedentary, benthic jellyfish that have invaded estuarine ecosystems around the world. Monitoring the spread of this invasive jellyfish must contend with high spatial and temporal variability in abundance of individuals, especially around their invasion front. Here, we evaluated the utility of drones to survey invasive Cassiopea in a coastal lake on the east coast of Australia. To assess the efficacy of a drone-based methodology, we compared the densities and counts of Cassiopea from drone observations to conventional boat-based observations and evaluated cost and time efficiency of these methods. We showed that there was no significant difference in Cassiopea density measured by drones compared to boat-based methods along the same transects. However, abundance estimates of Cassiopea derived from scaling-up transect densities were over-inflated by 319% for drones and 178% for boats, compared to drone-based counts of the whole site. Although conventional boat-based survey techniques were cost-efficient in the short-term, we recommend doing whole-of-site counts using drones. This is because it provides a time-saving and precise technique for long-term monitoring of the spatio-temporally dynamic invasion front of Cassiopea in coastal lakes and other sheltered marine habitats with relatively clear water.
Subject(s)
Behavior, Animal/physiology , Environmental Monitoring/methods , Unmanned Aerial Devices/ethics , Animals , Animals, Wild , Australia , Ecosystem , Environmental Monitoring/economics , Environmental Monitoring/instrumentation , Introduced Species/trends , Lakes , Scyphozoa/metabolism , WaterABSTRACT
Methyl-Coenzyme M Reductase (MCR) catalyzes the biosynthesis of methane in methanogenic archaea, using a catalytic Ni-centered Cofactor F430 in its active site. It also catalyzes the reverse reaction, that is, the anaerobic activation and oxidation, including the cleavage of the CH bond in methane. Because methanogenesis is the major source of methane on earth, understanding the reaction mechanism of this enzyme can have massive implications in global energy balances. While recent publications have proposed a radical-based catalytic mechanism as well as novel sulfonate-based binding modes of MCR for its native substrates, the structure of the active state of MCR, as well as a complete characterization of the reaction, remain elusive. Previous attempts to structurally characterize the active MCR-Ni(I) state have been unsuccessful due to oxidation of the redox- sensitive catalytic Ni center. Further, while many cryo structures of the inactive Ni(II)-enzyme in various substrates-bound forms have been published, no room temperature structures have been reported, and the structure and mechanism of MCR under physiologically relevant conditions is not known. In this study, we report the first room temperature structure of the MCRred1-silent Ni(II) form using an X-ray Free-Electron Laser (XFEL), with simultaneous X-ray Emission Spectroscopy (XES) and X-ray Diffraction (XRD) data collection. In celebration of the seminal contributions of inorganic chemist Dick Holm to our understanding of nickel-based catalysis, we are honored to announce our findings in this special issue dedicated to this remarkable pioneer of bioinorganic chemistry.
Subject(s)
Lasers , Methane , Crystallography, X-Ray , Methane/chemistry , Oxidation-Reduction , Oxidoreductases , TemperatureABSTRACT
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.
Subject(s)
Photosystem I Protein Complex/chemistry , Vitamin K 1/chemistry , Crystallography, X-Ray , Photosynthesis , Protein Structure, Tertiary , Temperature , ThermosynechococcusABSTRACT
A new species of the caprellid genus Paraproto, P. murrayae n. sp. is described based on specimens collected from New South Wales, Australia. The new species was collected from brown algae in shallow water (16-19 m deep). Paraproto murrayae n. sp. is very similar to P. tasmaniensis Guerra-García Takeuchi, 2004 but can be distinguished mainly by the following characteristics: (1) adults of P. murrayae are significantly smaller than P. tasmaniensis (5-6 mm and 10-11 mm respectively); (2) in larger males of P. tasmaniensis, gnathopod 2 is inserted on the anterior half of pereonite 2, rather than the posterior half as in P. murrayae; (3) the dactylus of the male gnathopod 2 is thickened medially in P. murrayae, but not thickened in P. tasmaniensis; (4) the setal formula of mandibular palp is 1-3-1 in P. murrayae versus 1-9-1 or 1-10-1 in P. tasmaniensis; (5) the lower lip is glabrous in P. murrayae but strongly setose in P. tasmaniensis; and (6) the anterolateral projections on pereonite 2 are lacking or vestigial in males of P. murrayae rather than distinct as in P. tasmaniensis. An illustrated key to the species of Paraproto is provided.
Subject(s)
Amphipoda , Animals , Australia , MaleABSTRACT
The biological reduction of dinitrogen (N2) to ammonia is catalyzed by the complex metalloenzyme nitrogenase. Structures of the nitrogenase component proteins, Iron (Fe) protein and Molybdenumiron (MoFe) protein, and the stabilized complexes these component proteins, have been determined, providing a foundation for a number of fundamental aspects of the complicated catalytic mechanism. The reduction of dinitrogen to ammonia is a complex process that involves the binding of N2 followed by reduction with multiple electrons and protons. Electron transfer into nitrogenase is typically constrained to the unique electron donor, the Fe protein. These constraints have prevented structural characterization of the active site with bound substrate. Recently it has been realized that selected amino acid substitutions in the environment of the active site metal cluster (Ironmolybdenum cofactor, FeMo-co) allow substrates to persist even in the resting state. Reported here is a 1.70Å crystal structure of a nitrogenase MoFe protein α-96ArgâGln variant with the alternative substrate acetylene trapped in a channel in close proximity to FeMo-co. Complementary theoretical calculations support the validity of the acetylene interaction at this site and is also consistent with more favorable interactions in the variant MoFe protein compared to the native MoFe protein. This work represents the first structural evidence of a substrate trapped in the nitrogenase MoFe protein and is consistent with earlier assignments of proposed substrate pathways and substrate binding sites deduced from biochemical, spectroscopic, and theoretical studies.
Subject(s)
Acetylene/chemistry , Iron/chemistry , Molybdenum/chemistry , Nitrogenase/chemistry , Catalytic Domain , Crystallography, X-Ray , Molecular Structure , Oxidation-Reduction , Substrate SpecificityABSTRACT
The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.
Subject(s)
Cadmium Compounds/chemistry , Molybdoferredoxin/chemistry , Nitrogen/chemistry , Nitrogenase/chemistry , Sulfides/chemistry , Adenosine Triphosphate/chemistry , Ammonia/chemistry , Catalysis/radiation effects , Hydrolysis/radiation effects , Light , Nanoparticles/chemistry , Nitrogen Fixation , Nitrogenase/radiation effects , Oxidation-Reduction/drug effects , Oxidation-Reduction/radiation effectsABSTRACT
We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Fe-S protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the X-ray crystal structure.
ABSTRACT
The X-ray crystal structure is presented for a nitrogenase MoFe protein where the alpha subunit residue at position 70 (alpha-70(Val)) has been substituted by the amino acid isoleucine (alpha-70(Ile)). Substitution of alpha-70(Val) by alpha-70(Ile) results in a MoFe protein that is hampered in its ability to reduce a range of substrates including acetylene and N(2), yet retains normal proton reduction activity. The 2.3A structure of the alpha-70(Ile) MoFe protein is compared to the alpha-70(Val) wild-type MoFe protein, revealing that the delta methyl group of alpha-70(Val) is positioned over Fe6 within the active site FeMo-cofactor. This work provides strong crystallographic support for the previously proposed model that substrates bind and are reduced at a single 4Fe-4S face of the FeMo-cofactor and that when alpha-70(Val) is substituted by alpha-70(Ile) access of substrates to Fe6 of this face is effectively blocked. Furthermore the detailed examination of the structure provides the basis for understanding the ability to trap and characterize hydrides in the variant, contributing significantly to our understanding of substrate access and substrate reduction at the FeMo-cofactor active site of nitrogenase.