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Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe 8 S 7 ]) and M-cluster core (or L-cluster; [Fe 8 S 9 C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD , nifK , nifH , nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB , nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli . Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.

Determining the requirements for efficient oxygen (O 2 ) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O 2 ) and a sequential mechanism (cofactor binding and reaction with O 2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the Fe II ), αKG binding to Fe II -DAOCS results in ∼45% five-coordinate sites that selectively react with O 2 relative to the remaining six-coordinate sites. However, this reaction produces an Fe III species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to Fe II -DAOCS produces five-coordinate sites that rapidly react with O 2 to form an Fe IV =O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes., Competing Interests: The authors declare no competing interest.

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to C α -formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O 2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O 2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation., Competing Interests: Conflict of interest statement: C.R.B. is a cofounder and member of the Scientific Advisory Board of Redwood Bioscience (a subsidiary of Catalent, Inc.), which has exclusive rights to the SMARTag technology based on protein modification by FGE. C.R.B. is also a cofounder of Palleon Pharmaceuticals, Enable Biosciences, and InterVenn Biosciences, and a member of the Board of Directors of Eli Lilly & Co.

Hemoglobin and myoglobin are oxygen-binding proteins with S = 0 heme {FeO 2 } 8 active sites. The electronic structure of these sites has been the subject of much debate. This study utilizes Fe K-edge X-ray absorption spectroscopy (XAS) and 1s2p resonant inelastic X-ray scattering (RIXS) to study oxyhemoglobin and a related heme {FeO 2 } 8 model compound, [(pfp)Fe(1-MeIm)(O 2 )] (pfp = meso-tetra(α,α,α,α- o -pivalamido-phenyl)porphyrin, or TpivPP, 1-MeIm = 1-methylimidazole) (pfpO 2 ), which was previously analyzed using L-edge XAS. The K-edge XAS and RIXS data of pfpO 2 and oxyhemoglobin are compared with the data for low-spin Fe II and Fe III [Fe(tpp)(Im) 2 ] 0/+ (tpp = tetra-phenyl porphyrin) compounds, which serve as heme references. The X-ray data show that pfpO 2 is similar to Fe II , while oxyhemoglobin is qualitatively similar to Fe III , but with significant quantitative differences. Density-functional theory (DFT) calculations show that the difference between pfpO 2 and oxyhemoglobin is due to a distal histidine H bond to O 2 and the less hydrophobic environment in the protein, which lead to more backbonding into the O 2 A valence bond configuration interaction multiplet model is used to analyze the RIXS data and show that pfpO 2 is dominantly Fe II with 6-8% Fe III character, while oxyhemoglobin has a very mixed wave function that has 50-77% Fe III character and a partially polarized Fe-O 2 π-bond., Competing Interests: The authors declare no conflict of interest.

A direct, catalytic conversion of benzene to phenol would have wide-reaching economic impacts. Fe zeolites exhibit a remarkable combination of high activity and selectivity in this conversion, leading to their past implementation at the pilot plant level. There were, however, issues related to catalyst deactivation for this process. Mechanistic insight could resolve these issues, and also provide a blueprint for achieving high performance in selective oxidation catalysis. Recently, we demonstrated that the active site of selective hydrocarbon oxidation in Fe zeolites, named α-O, is an unusually reactive Fe(IV)=O species. Here, we apply advanced spectroscopic techniques to determine that the reaction of this Fe(IV)=O intermediate with benzene in fact regenerates the reduced Fe(II) active site, enabling catalytic turnover. At the same time, a small fraction of Fe(III)-phenolate poisoned active sites form, defining a mechanism for catalyst deactivation. Density-functional theory calculations provide further insight into the experimentally defined mechanism. The extreme reactivity of α-O significantly tunes down (eliminates) the rate-limiting barrier for aromatic hydroxylation, leading to a diffusion-limited reaction coordinate. This favors hydroxylation of the rapidly diffusing benzene substrate over the slowly diffusing (but more reactive) oxygenated product, thereby enhancing selectivity. This defines a mechanism to simultaneously attain high activity (conversion) and selectivity, enabling the efficient oxidative upgrading of inert hydrocarbon substrates., Competing Interests: The authors declare no conflict of interest.

Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N 2 O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. Density functional theory calculations clarify how the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction., Competing Interests: The authors declare no conflict of interest.

NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster-bound NifEN (designated NifEN(M)) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifEN(M) upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN(M) with an M-cluster in one αβ-half and an empty cluster-binding site in the other αβ-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αβ-dimers of NifEN. Perhaps most importantly, NifEN(M) displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αβ-dimer of NifEN(M) as a structural and functional mimic of one catalytic αβ-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase., Competing Interests: The authors declare no conflict of interest.

Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.

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 β2-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.

Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.

Hemoglobin (Hb) is the heme-containing O2 transport protein essential for life in all vertebrates. The resting high-spin (S = 2) ferrous form, deoxy-Hb, combines with triplet O2, forming diamagnetic (S = 0) oxy-Hb. Understanding this electronic structure is the key first step in understanding transition metal-O2 interaction. However, despite intense spectroscopic and theoretical studies, the electronic structure description of oxy-Hb remains elusive, with at least three different descriptions proposed by Pauling, Weiss, and McClure-Goddard, based on theory, spectroscopy, and crystallography. Here, a combination of X-ray absorption spectroscopy and extended X-ray absorption fine structure, supported by density functional theory calculations, help resolve this debate. X-ray absorption spectroscopy data on solution and crystalline oxy-Hb indicate both geometric and electronic structure differences suggesting that two of the previous descriptions are correct for the Fe-O2 center in oxy-Hb. These results support the multiconfigurational nature of the ground state developed by theoretical results. Additionally, it is shown here that small differences in hydrogen bonding and solvation effects can tune the ground state, tipping it into one of the two probable configurations. These data underscore the importance of solution spectroscopy and show that the electronic structure in the crystalline form may not always reflect the true ground-state description in solution.

Bleomycin (BLM) is a glycopeptide anticancer drug capable of effecting single- and double-strand DNA cleavage. The last detectable intermediate prior to DNA cleavage is a low spin Fe(III) peroxy level species, termed activated bleomycin (ABLM). DNA strand scission is initiated through the abstraction of the C-4' hydrogen atom of the deoxyribose sugar unit. Nuclear resonance vibrational spectroscopy (NRVS) aided by extended X-ray absorption fine structure spectroscopy and density functional theory (DFT) calculations are applied to define the natures of Fe(III)BLM and ABLM as (BLM)Fe(III)─OH and (BLM)Fe(III)(η(1)─OOH) species, respectively. The NRVS spectra of Fe(III)BLM and ABLM are strikingly different because in ABLM the δFe─O─O bending mode mixes with, and energetically splits, the doubly degenerate, intense O─Fe─N(ax) transaxial bends. DFT calculations of the reaction of ABLM with DNA, based on the species defined by the NRVS data, show that the direct H-atom abstraction by ABLM is thermodynamically favored over other proposed reaction pathways.

The P-cluster of nitrogenase is one of the most complex biological metallocenters known to date. Despite the recent advances in the chemical synthesis of P-cluster topologs, the biosynthetic mechanism of P-cluster has not been well defined. Here, we present a combined biochemical, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine-structure investigation of the maturation process of P-clusters in DeltanifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe(4)S(4)]-like cluster pairs in DeltanifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [Fe(8)S(7)] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in DeltanifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the alpha(2)beta(2)-tetrameric MoFe protein, i.e., one P-cluster is formed in one alphabeta dimer before the other in the second alphabeta dimer.

Multicopper oxidases (MCOs) catalyze the 4e(-) reduction of O(2) to H(2)O. The reaction of the fully reduced enzyme with O(2) generates the native intermediate (NI), which undergoes a slow decay to the resting enzyme in the absence of substrate. NI is a fully oxidized form, but its spectral features are very different from those of the resting form (also fully oxidized), because the type 2 and the coupled-binuclear type 3 Cu centers in the O(2)-reducing trinuclear Cu cluster site are isolated in the resting enzyme, whereas these are all bridged by a micro(3)-oxo ligand in NI. Notably, the one azide-bound NI (NI(Az)) exhibits spectral features very similar to those of NI, in which the micro(3)-oxo ligand in NI has been replaced by a micro(3)-bridged azide. Comparison of the spectral features of NI and NI(Az), combined with density functional theory (DFT) calculations, allows refinement of the NI structure. The decay of NI to the resting enzyme proceeds via successive proton-assisted steps, whereas the rate-limiting step involves structural rearrangement of the micro(3)-oxo-bridge from inside to outside the cluster. This phenomenon is consistent with the slow rate of NI decay that uncouples the resting enzyme from the catalytic cycle, leaving NI as the catalytically relevant fully oxidized form of the MCO active site. The all-bridged structure of NI would facilitate electron transfer to all three Cu centers of the trinuclear cluster for rapid proton-coupled reduction of NI to the fully reduced form for catalytic turnover.

The Fe protein is indispensable for nitrogenase catalysis and biosynthesis. However, its function in iron-molybdenum cofactor (FeMoco) biosynthesis has not been clearly defined. Here we show that the Fe protein can act as a Mo/homocitrate insertase that mobilizes Mo/homocitrate for the maturation of FeMoco precursor on NifEN. Further, we establish that Mo/homocitrate mobilization by the Fe protein likely involves hydrolysis of MgATP and protein-protein interaction between the Fe protein and NifEN. Our findings not only clarify the role of the Fe protein in FeMoco assembly and assign another function to this multitask enzyme but also provide useful insights into a mechanism of metal trafficking required for the assembly of complex metalloproteins such as nitrogenase.

FeMo cofactor (FeMoco) biosynthesis is one of the most complicated processes in metalloprotein biochemistry. Here we show that Mo and homocitrate are incorporated into the Fe/S core of the FeMoco precursor while it is bound to NifEN and that the resulting fully complemented, FeMoco-like cluster is transformed into a mature FeMoco upon transfer from NifEN to MoFe protein through direct protein-protein interaction. Our findings not only clarify the process of FeMoco maturation, but also provide useful insights into the other facets of nitrogenase chemistry.

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron- and sulfur-containing FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe-4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco.

Nitrogenase is a multicomponent metalloenzyme that catalyzes the conversion of atmospheric dinitrogen to ammonia. For decades, it has been generally believed that the [8Fe-7S] P-cluster of nitrogenase component 1 is indispensable for nitrogenase activity. In this study, we identified two catalytically active P-cluster variants by activity assays, metal analysis, and EPR spectroscopic studies. Further, we showed that both P-cluster variants resemble [4Fe-4S]-like centers based on x-ray absorption spectroscopic experiments. We believe that our findings challenge the dogma that the standard P-cluster is the only cluster species capable of supporting substrate reduction at the FeMo cofactor and provide important insights into the general mechanism of nitrogenase catalysis and assembly.

We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 A. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining single-particle diffraction with x-ray free electron lasers.

Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

We describe an approach to the high-resolution three-dimensional structural determination of macromolecules that utilizes ultrashort, intense x-ray pulses to record diffraction data in combination with direct phase retrieval by the oversampling technique. It is shown that a simulated molecular diffraction pattern at 2.5-A resolution accumulated from multiple copies of single rubisco biomolecules, each generated by a femtosecond-level x-ray free electron laser pulse, can be successfully phased and transformed into an accurate electron density map comparable to that obtained by more conventional methods. The phase problem is solved by using an iterative algorithm with a random phase set as an initial input. The convergence speed of the algorithm is reasonably fast, typically around a few hundred iterations. This approach and phasing method do not require any ab initio information about the molecule, do not require an extended ordered lattice array, and can tolerate high noise and some missing intensity data at the center of the diffraction pattern. With the prospects of the x-ray free electron lasers, this approach could provide a major new opportunity for the high-resolution three-dimensional structure determination of single biomolecules.

The biological N2-fixation reaction is catalyzed by the enzyme nitrogenase. The metal cluster active site of this enzyme, the iron-molybdenum cofactor (FeMoco), can be studied either while bound within the MoFe protein component of nitrogenase or after it has been extracted into N-methylformamide. The two species are similar but not identical. For example, the addition of thiophenol or selenophenol to isolated FeMoco causes its rather broad S = 3/2 electron paramagnetic resonance signal to sharpen and more closely approach the signal exhibited by protein-bound FeMoco. The nature of this thiol/selenol binding site has been investigated by using Se-K edge extended x-ray absorption fine structure (EXAFS) to study selenophenol ligated to FeMoco, and the results are reported here. EXAFS data analysis at the ligand Se-K edge was performed with a set of software, GNXAS, that provides for direct calculation of the theoretical EXAFS signals and least-squares fits to the experimental data. Data analysis results show definitively that the selenol (and by inference thiol) binds to Fe at a distance of 2.4 A. In contrast, unacceptable fits are obtained with either Mo or S as the liganded atom (instead of Fe). These results provide quantitative details about an exchangeable thiol/selenol binding site on FeMoco in its isolated, solution state and establish an Fe atom as the site of this reaction. Furthermore, the utility of ligand-based EXAFS as a probe of coordination in polynuclear metal clusters is demonstrated.

The nickel/iron/sulfur center of the carbon monoxide dehydrogenase (carbon monoxide:(acceptor)oxidoreductase; EC 1.2.99.2) enzyme from Rhodospirillum rubrum (Rr-CODH) was studied by x-ray absorption spectroscopy at the Ni K edge. Extended x-ray absorption fine structure data show that the first Ni coordination shell consists of 2 S atoms at 2.23 A and 2-3 N/O atoms at 1.87 A. The edge structure indicates a distorted tetrahedral or five-coordinate Ni environment in both oxidized and reduced Rr-CODH. By comparing second-shell extended x-ray absorption fine structure data of Rr-CODH to that of (Et4N)3[NiFe3S4(SEt)4], a cubane-type cluster, it was clearly established that Ni in the Rr-CODH center is not involved in the core of a NiFe3S4 cubane cluster. One model consistent with the results is a mononuclear Ni2+ site, bridged by S-Cys or sulfide to one or both of the Fe4S4 clusters of the enzyme, with the remaining coordination sites occupied by additional S-Cys or N/O-liganding amino acid residues.

The technique of x-ray absorption spectroscopy using tuneable, very intense x-rays from a high energy electron storage ring has been applied to study of the estended x-ray absorption fine structure for Cu and Ni tetraphenylporphyrin and methemoglobin. Preliminary analysis shows that the spectra may be interpreted as a super-position of modulations arising from the nearest neighbor nitrogen and pyrrole alpha-carbon coordination sheels of the metal atoms. We estimate that with the observed magnitude of noise to modulation amplitude, relative shifts of 0,5% in the metal-nitrogen to metal-carbon bond distances in the prophyrins should be observable using extended x-ray absorption fine structure and that this technique may provide a method of observing these types of structural changes in solution.

The primary coordination environment of the "blue" copper ion in oxidized azurin has been elucidated by x-ray absorption spectroscopy. The most striking feature is the unambiguous presence of a very short copper-sulfur distance at 2.10 +/- 0.02 A. Nitrogen ligands, presumed to be from imidazoles, are found at 1.97 A. There is some evidence that the copper coordination sphere may be completed by a second sulfur, the distance of which is determined with much less certainty.

X-ray absorption spectroscopy has been used to study the dimeric iron center in azidomethemerythrin from Phascolopsis gouldii. Absorption edge data confirm that the two iron atoms are present as Fe(III) and suggest a hexa-coordination site for each of the iron atoms. The extended x-ray absorption fine structure (EXAFS) analysis provides direct structural evidence of a mu-oxo bridge between the two iron atoms at an average Fe-O distance of 1.71-1.76 A. Analysis using a multiple-scattering formalism calculates upper limits of 165 degrees for the Fe-O-Fe bridging angle and 3.38 A for the Fe-Fe distance. This result agrees with current crystallographic models being determined by refinement of structures of two azidomethemerythrins.

Crystals of monellin, a sweet protein from Dioscoreophyllum cumminsii, were grown by vapor diffusion of 20% ethanol into buffered protein solution. The crystals are orthorhombic, belonging to space group P2(1)2(1)2, with a = 54.4 A, b = 113.0 A, c = 40.8 A, and V = 250,300 A(3). The asymmetric unit contains two complete molecules of monellin. The diffraction pattern of this crystal form extends to at least 2.5 A, indicating that x-ray structural analysis is possible to near-atomic resolution.

By using ultra-high-flux synchrotron x-radiation from a wiggler source, good Laue diffraction data have been obtained from protein microcrystals of size 30 X 35 X 10 microns3, mounted wet in glass capillaries. At the flux level of 10(13)-10(14) photons per sec/mm2, the radiation damage is still low enough to allow a large survey of reciprocal space for a microcrystal and a complete survey for a normal-sized protein crystal. The development of sources for ultra-high-intensity synchrotron radiation is thus an important improvement in the technique for determination of structure through protein crystallography as well as in other cases where crystal size is often a limiting factor.

X-ray diffraction photographs of protein single crystals have been obtained using synchrotron radiation produced by an electron-positron storage ring. The diffracted intensities observed with this unconventional source are a factor of at least 60 greater than those obtained with a sealed x-ray tube using the same crystal and instrumental parameters. Diffraction data have been collected by the precession method to higher resolution and using smaller protein crystals than would have been possible with a conventional source. The crystal decay rate in the synchrotron beam for several proteins appears to be substantially less than that observed with Ni-filtered Cu radiation. The tunable nature of the source (which allows selective optimization of anomalous contributions to the scattering factors) and the low angular divergence of the beam make the source very useful for single crystal protein diffraction studies.

Beef heart aconitase contains 3Fe clusters in its inactive and 4Fe clusters in its active form. The fully active form can be restored from the inactive one by insertion of Fe(2+), whereas S(2-) is not required. Chemical analyses for iron and labile sulfide yield Fe/S(2-) ratios of 0.66-0.74 for the inactive and 0.90-1.03 for the active form. Sulfane sulfur (S(0)) was not detected. We propose on the basis of these data that the inactive form may arise from the active one by loss of one iron only per cluster with the sulfur remaining as S(2-) in a [3Fe-4S] structure. Measurements by extended x-ray absorption fine structure (EXAFS) spectroscopy on the 3Fe form of aconitase yield a Fe..S distance of 2.24 A and a Fe..Fe distance of 2.71 A. This Fe..Fe distance is in agreement with that obtained by EXAFS on ferredoxin II of Desulfovibrio gigas, another 3Fe protein, but disagrees with Fe..Fe distances observed for the 3Fe cluster of Azotobacter vinelandii ferredoxin I by x-ray diffraction-namely, 4.1 A. We suggest that this difference may be due to the presence of a [3Fe-3S] structure in the Azotobacter ferredoxin I crystals vs. a [3Fe-4S] structure in liquid or frozen solutions of aconitase. The [3Fe-3S] cluster has been shown to have a relatively flat twist-boat structure, whereas a [3Fe-4S] cluster could be expected to essentially maintain the compact structure of the [4Fe-4S] cluster. This would explain the differences in Fe..Fe distances. Two possible structural models for a [3Fe-4S] cluster are discussed.

We have measured high-resolution x-ray absorption spectra of lanthanide (Ln) and heavy transition metal complexes that display prominent narrow absorption peaks near the L2 and L3 absorption edges. The anomalous scattering factors (f' and f"), which are mathematically related to the absorption cross section, have correspondingly sharp changes in their magnitude within 5-10 eV of the absorption edge. Calculations of the magnitude of the change in f' and f" demonstrate that significant changes (on the order of 20 electrons in f') can be expected for these materials. These substantial changes in the anomalous scattering factors have applications to deriving structural information for macromolecules from x-ray diffraction studies. The magnitude of the changes indicate that the anomalous scattering technique is a powerful means of obtaining structural characteristics for macromolecules in single crystals, in solution, and in biological membranes.

Fe(CN)6(-3) oxidation of the aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I, (7Fe)FdI, is a degradative reaction. Destruction of the [4Fe-4S] cluster occurs first, followed by destruction of the [3Fe-3S] cluster. At a Fe(CN)6(-3)/(7Fe)FdI concentration ratio of 20, the product is a mixture of apoprotein and protein containing only a [3Fe-3S] cluster, (3Fe)FdI. This protein mixture, after partial purification, has been characterized by absorption, CD, magnetic CD, and EPR and Fe x-ray absorption spectroscopies. EPR and magnetic CD spectra provide strong evidence that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in (7Fe)FdI. Analysis of the extended x-ray absorption fine structure (EXAFS) of (3Fe)FdI finds Fe scattering at an average Fe...Fe distance of approximately equal to 2.7 A. The structure of the oxidized [3Fe-3S] cluster in solutions of oxidized (3Fe)FdI, and, by extension, of oxidized (7Fe)FdI, is thus different from that obtained by x-ray crystallography on oxidized (7Fe)FdI. Possible interpretations of this result are discussed.

Crystals of the nerve growth factor protein were grown by vapor diffusion from ethanol solution. The crystals are hexagonal, belonging to space group P622 (or its enantiomorph) with a equals 56.1 A, c equals 181.4 A, and V equals 494,400 A. The unit cell contains six molecules of dimeric protein and thus has one monomer per asymmetric unit. The diffraction pattern extends to at least 2.7 A, indicating that this crystal form is suitable for structural analysis to near-atomic resolution.