Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin.

In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (E°') of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) from Pseudomonas aeruginosa. These variants were found to differentially influence the E°' of T1Cu, with M13F Az decreasing E°', M44F Az increasing E°', and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increases E°' by 26 mV relative to WT-Az, which is very close to the combined effect of E°' by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.

Electron Paramagnetic Resonance and Synchrotron X-ray Absorption Spectroscopy for Highly Sensitive Characterization of Calcium Arsenates.

Calcium arsenates such as pharmacolite (CaHAsO4·2H2O), haidingerite (CaHAsO4·H2O), and weilite (CaHAsO4) are important sinks for arsenic in mine tailings as well as other natural and contaminated sites and are useful for reducing the mobility and bioavailability of this toxic metalloid in the environment. However, calcium arsenates usually occur in trace amounts dominated by other phases, making their detection, identification, and quantification challenging. In this contribution, pharmacolite, haidingerite, and weilite are shown to exhibit subtle but distinct postedge differences in As K-edge X-ray absorption near-edge structure (XANES) spectra and feature characteristic [AsO3]2-, [AsO4]2-, and [AsO4]4- radicals, all derived from the diamagnetic [HAsO4]2- precursor during γ-ray irradiation, in electron paramagnetic resonance (EPR) spectra. In particular, the 75As (nuclear spin I = 3/2 and natural isotope abundance = 100%) hyperfine coupling constants of the [AsO3]2- radicals in pharmacolite and haidingerite as well as other minerals (e.g., calcite and gypsum) are clearly distinct, allowing the unambiguous identification of calcium arsenates by the EPR technique readily at ∼0.1 wt %. Similarly, linear combination fittings of As K-edge XANES spectra demonstrate that pharmacolite and haidingerite at ∼0.1 wt % each in gypsum-rich mixtures can be detected and quantified as well. Therefore, a combination of the EPR and XANES techniques is a powerful approach for the highly sensitive characterization of calcium arsenates in the quest for the safe management and remediation of arsenic contamination. This work demonstrates the highly sensitive characterization of calcium arsenates by integrated electron paramagnetic resonance and synchrotron X-ray absorption spectroscopy.

Structural Basis for a Quadratic Relationship between Electronic Absorption and Electronic Paramagnetic Resonance Parameters of Type 1 Copper Proteins.

Type 1 copper (T1Cu) proteins play important roles in electron transfer in biology, largely due to the unique structure of the T1Cu center, which is reflected by its spectroscopic properties. Previous reports have suggested a correlation between a high ratio of electronic absorbance at ∼450 nm to that at ∼600 nm (R = A450/A600) and a large copper(II) hyperfine coupling in the z direction (Az) in electron paramagnetic resonance (EPR). However, this correlation does not have a clear physical meaning, nor does it hold for many proteins with a perturbed T1Cu center. To address this issue, a new parameter of R' [A450/(A450 + A600)] with a better physical meaning of a fractional SCys pseudo-σ to Cu(II) charge transfer transition intensity is defined and a quadratic relationship between R' and Az is found on the basis of a comprehensive analysis of ultraviolet-visible absorption, EPR, and structural parameters of T1Cu proteins. We are able to find good correlations between R' and the displacement of copper from the trigonal plane defined by the His2Cys ligands and the angle between the NHis1-Cu-NHis2 plane and the SCys-Cu-axial ligand plane, providing a structural basis for the observed correlation. These findings and analyses provide a new framework for a deeper understanding of the spectroscopic and electronic properties of T1Cu proteins, which may allow better design and applications of this important class of proteins for redox and electron transfer functions.

Sequestration of Selenite and Selenate in Gypsum (CaSO4·2H2O): Insights from the Single-Crystal Electron Paramagnetic Resonance Spectroscopy and Synchrotron X-ray Absorption Spectroscopy Study.

Gypsum is the most common sulfate mineral on Earth's surface and is the dominant solid byproduct in a wide variety of mining and industrial processes, thus representing a major source for heavy metal(loid) contamination, including selenium. Gypsum crystals grown from the gel diffusion technique in 0.02 M Na2SeO4 solution at pH 7.5 and 0.02 M Na2SeO3 solutions at pH 7.5 and 9.0 contain 828, 5198, and 5955 ppm Se, respectively. Synchrotron Se K-edge X-ray absorption spectroscopic analyses show that selenite and selenate are the dominant species in Se4+- and Se6+-doped gypsum, respectively. The single-crystal EPR spectra of Se4+- and Se6+-doped gypsum after gamma-ray irradiation reveal five selenium-centered oxyradicals: SeO2-(I), SeO2-(II), SeO2-(III), SeO3-, and HSeO42-. The former three radicals provide unequivocal evidence for the substitution of their paramagnetic precursor SeO32- for SO42- in the gypsum structure, while the latter two confirm the replacement of SeO42- for SO42-. These results demonstrate that gypsum has a significant capacity for sequestrating both selenite and selenate in the structure but has a marked preference for the former, thus confirming important controls on the mobility and bioavailability of selenium oxyanions and pointing to optimal applications of gypsum for remediating selenium contamination under neutral to alkaline conditions.

Design of a single protein that spans the entire 2-V range of physiological redox potentials.

The reduction potential (E°') is a critical parameter in determining the efficiency of most biological and chemical reactions. Biology employs three classes of metalloproteins to cover the majority of the 2-V range of physiological E°'s. An ultimate test of our understanding of E°' is to find out the minimal number of proteins and their variants that can cover this entire range and the structural features responsible for the extreme E°'. We report herein the design of the protein azurin to cover a range from +970 mV to -954 mV vs. standard hydrogen electrode (SHE) by mutating only five residues and using two metal ions. Spectroscopic methods have revealed geometric parameters important for the high E°'. The knowledge gained and the resulting water-soluble redox agents with predictable E°'s, in the same scaffold with the same surface properties, will find wide applications in chemical, biochemical, biophysical, and biotechnological fields.

Near-Infrared Photoactivatable Nitric Oxide Donors with Integrated Photoacoustic Monitoring.

Photoacoustic (PA) tomography is a noninvasive technology that utilizes near-infrared (NIR) excitation and ultrasonic detection to image biological tissue at centimeter depths. While several activatable small-molecule PA sensors have been developed for various analytes, the use of PA molecules for deep-tissue analyte delivery and monitoring remains an underexplored area of research. Herein, we describe the synthesis, characterization, and in vivo validation of photoNOD-1 and photoNOD-2, the first organic, NIR-photocontrolled nitric oxide (NO) donors that incorporate a PA readout of analyte release. These molecules consist of an aza-BODIPY dye appended with an aryl N-nitrosamine NO-donating moiety. The photoNODs exhibit chemostability to various biological stimuli, including redox-active metals and CYP450 enzymes, and demonstrate negligible cytotoxicity in the absence of irradiation. Upon single-photon NIR irradiation, photoNOD-1 and photoNOD-2 release NO as well as rNOD-1 or rNOD-2, PA-active products that enable ratiometric monitoring of NO release. Our in vitro studies show that, upon irradiation, photoNOD-1 and photoNOD-2 exhibit 46.6-fold and 21.5-fold ratiometric turn-ons, respectively. Moreover, unlike existing NIR NO donors, the photoNODs do not require encapsulation or multiphoton activation for use in live animals. In this study, we use PA tomography to monitor the local, irradiation-dependent release of NO from photoNOD-1 and photoNOD-2 in mice after subcutaneous treatment. In addition, we use a murine model for breast cancer to show that photoNOD-1 can selectively affect tumor growth rates in the presence of NIR light stimulation following systemic administration.

Copper-sulfenate complex from oxidation of a cavity mutant of Pseudomonas aeruginosa azurin.

Metal-sulfenate centers are known to play important roles in biology and yet only limited examples are known due to their instability and high reactivity. Herein we report a copper-sulfenate complex characterized in a protein environment, formed at the active site of a cavity mutant of an electron transfer protein, type 1 blue copper azurin. Reaction of hydrogen peroxide with Cu(I)-M121G azurin resulted in a species with strong visible absorptions at 350 and 452 nm and a relatively low electron paramagnetic resonance gz value of 2.169 in comparison with other normal type 2 copper centers. The presence of a side-on copper-sulfenate species is supported by resonance Raman spectroscopy, electrospray mass spectrometry using isotopically enriched hydrogen peroxide, and density functional theory calculations correlated to the experimental data. In contrast, the reaction with Cu(II)-M121G or Zn(II)-M121G azurin under the same conditions did not result in Cys oxidation or copper-sulfenate formation. Structural and computational studies strongly suggest that the secondary coordination sphere noncovalent interactions are critical in stabilizing this highly reactive species, which can further react with oxygen to form a sulfinate and then a sulfonate species, as demonstrated by mass spectrometry. Engineering the electron transfer protein azurin into an active copper enzyme that forms a copper-sulfenate center and demonstrating the importance of noncovalent secondary sphere interactions in stabilizing it constitute important contributions toward the understanding of metal-sulfenate species in biological systems.

Mechanism of H2 Production by Models for the [NiFe]-Hydrogenases: Role of Reduced Hydrides.

The intermediacy of a reduced nickel-iron hydride in hydrogen evolution catalyzed by Ni-Fe complexes was verified experimentally and computationally. In addition to catalyzing hydrogen evolution, the highly basic and bulky (dppv)Ni(µ-pdt)Fe(CO)(dppv) ([1](0); dppv = cis-C2H2(PPh2)2) and its hydride derivatives have yielded to detailed characterization in terms of spectroscopy, bonding, and reactivity. The protonation of [1](0) initially produces unsym-[H1](+), which converts by a first-order pathway to sym-[H1](+). These species have C1 (unsym) and Cs (sym) symmetries, respectively, depending on the stereochemistry of the octahedral Fe site. Both experimental and computational studies show that [H1](+) protonates at sulfur. The S = 1/2 hydride [H1](0) was generated by reduction of [H1](+) with Cp*2Co. Density functional theory (DFT) calculations indicate that [H1](0) is best described as a Ni(I)-Fe(II) derivative with significant spin density on Ni and some delocalization on S and Fe. EPR spectroscopy reveals both kinetic and thermodynamic isomers of [H1](0). Whereas [H1](+) does not evolve H2 upon protonation, treatment of [H1](0) with acids gives H2. The redox state of the "remote" metal (Ni) modulates the hydridic character of the Fe(II)-H center. As supported by DFT calculations, H2 evolution proceeds either directly from [H1](0) and external acid or from protonation of the Fe-H bond in [H1](0) to give a labile dihydrogen complex. Stoichiometric tests indicate that protonation-induced hydrogen evolution from [H1](0) initially produces [1](+), which is reduced by [H1](0). Our results reconcile the required reductive activation of a metal hydride and the resistance of metal hydrides toward reduction. This dichotomy is resolved by reduction of the remote (non-hydride) metal of the bimetallic unit.

A Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear Type 1 Copper Center with an Open Binding Site.

Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M(-1) cm(-1)) and 558 nm (2290 M(-1) cm(-1)) in the UV-vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10(-4) cm(-1), and another more rhombic, with a smaller A∥ value of 69 × 10(-4) cm(-1). The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV-vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.

Binuclear Cu(A) Formation in Biosynthetic Models of Cu(A) in Azurin Proceeds via a Novel Cu(Cys)2His Mononuclear Copper Intermediate.

Cu(A) is a binuclear electron transfer (ET) center found in cytochrome c oxidases (CcOs), nitrous oxide reductases (N2ORs), and nitric oxide reductase (NOR). In these proteins, the Cu(A) centers facilitate efficient ET (kET > 104s⁻¹) under low thermodynamic driving forces (10-90 mV). While the structure and functional properties of Cu(A) are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu(A) maturation process are still lacking. Previous studies of the Cu(A) assembly mechanism in vitro using a biosynthetic model Cu(A) center in azurin (Cu(A)Az) identified a novel intermediate X (Ix) during reconstitution of the binuclear site. However, because of the instability of Ix and the coexistence of other Cu centers, such as Cu(A)' and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of Cu(A) assembly using variants of Glu114XCuAAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu(A) site, with a major focus on characterization of the novel intermediate Ix. We show that Cu(A) assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate Ix, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → Ix formation, suggesting that Cu(A) formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained Ix in ∼80-85% population in these variants, which allowed us to employ ultraviolet-visible, electron paramagnetic resonance, and extended X-ray absorption fine structure spectroscopic techniques to identify the Ix as a mononuclear Cu(Cys)(2)(His) complex. Because some of the intermediates have been proposed to be involved in the assembly of native Cu(A), these results shed light on the structural features of the important intermediates and mechanism of Cu(A) formation.

Defining the role of tyrosine and rational tuning of oxidase activity by genetic incorporation of unnatural tyrosine analogs.

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.

Rationally tuning the reduction potential of a single cupredoxin beyond the natural range.

Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer in photosynthesis and respiration to catalysis in industrial and fuel cell research. These functions are accomplished in nature by only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or electron-transfer properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centres or fuel cell catalysts for energy conversion. Here we show that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of azurin is now predictable across the full range of cupredoxin potentials.

ESR and X-ray Structure Investigations on the Binding and Mechanism of Inhibition of the Native State of Myeloperoxidase with Low Molecular Weight Fragments.

As an early visitor to the injured loci, neutrophil-derived human Myeloperoxidase (hMPO) offers an attractive protein target to modulate the inflammation of the host tissue through suitable inhibitors. We describe a novel methodology of using low temperature ESR spectroscopy (6 K) and FAST™ technology to screen a diverse series of small molecules that inhibit the peroxidase function through reversible binding to the native state of MPO. Our initial efforts to profile molecules on the inhibition of MPO-initiated nitration of the Apo-A1 peptide (AEYHAKATEHL) assay showed several potent (with sub-micro molar IC50s) but spurious inhibitors that either do not bind to the heme pocket in the enzyme or retain high (>50 %) anti oxidant potential. Such molecules when taken forward for X-ray did not yield inhibitor-bound co-crystals. We then used ESR to confirm direct binding to the native state enzyme, by measuring the binding-induced shift in the electronic parameter g to rank order the molecules. Molecules with a higher rank order-those with g-shift Rrelative ≥15-yielded well-formed protein-bound crystals (n = 33 structures). The co-crystal structure with the LSN217331 inhibitor reveals that the chlorophenyl group projects away from the heme along the edges of the Phe366 and Phe407 side chain phenyl rings thereby sterically restricting the access to the heme by the substrates like H2O2. Both ESR and antioxidant screens were used to derive the mechanism of action (reversibility, competitive substrate inhibition, and percent antioxidant potential). In conclusion, our results point to a viable path forward to target the native state of MPO to tame local inflammation.

Direct EPR observation of a tyrosyl radical in a functional oxidase model in myoglobin during both H2O2 and O2 reactions.

Tyrosine is a conserved redox-active amino acid that plays important roles in heme-copper oxidases (HCO). Despite the widely proposed mechanism that involves a tyrosyl radical, its direct observation under O2 reduction conditions remains elusive. Using a functional oxidase model in myoglobin called F33Y-Cu(B)Mb that contains an engineered tyrosine, we report herein direct observation of a tyrosyl radical during both reactions of H2O2 with oxidized protein and O2 with reduced protein by electron paramagnetic resonance spectroscopy, providing a firm support for the tyrosyl radical in the HCO enzymatic mechanism.

Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein: preparation and study of Fe(II)-M121E azurin.

Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.

Spectroscopic and computational study of a nonheme iron nitrosyl center in a biosynthetic model of nitric oxide reductase.

A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of a selective probe of NO binding to the nonheme FeB center. By replacing the heme in a biosynthetic model of NORs, which structurally and functionally mimics NORs, with isostructural ZnPP, the electronic structure and functional properties of the FeB nitrosyl complex was probed. This approach allowed observation of the first S=3/2 nonheme {FeNO}(7) complex in a protein-based model system of NOR. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}(7) complex is best described as a high spin ferrous iron (S=2) antiferromagnetically coupled to an NO radical (S=1/2) [Fe(2+)-NO(.)]. The radical nature of the FeB -bound NO would facilitate N-N bond formation by radical coupling with the heme-bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs.

Isolation of a mixed valence diiron hydride: evidence for a spectator hydride in hydrogen evolution catalysis.

The mixed-valence diiron hydrido complex (µ-H)Fe2(pdt)(CO)2(dppv)2 ([H1](0), where pdt =1,3-propanedithiolate and dppv = cis-1,2-C2H2(PPh2)2), was generated by reduction of the differous hydride [H1](+) using decamethylcobaltocene. Crystallographic analysis shows that [H1](0) retains the stereochemistry of its precursor, where one dppv ligand spans two basal sites and the other spans apical and basal positions. The Fe---Fe bond elongates to 2.80 from 2.66 Å, but the Fe-P bonds only change subtly. Although the Fe-H distances are indistinguishable in the precursor, they differ by 0.2 Å in [H1](0). The X-band electron paramagnetic resonance (EPR) spectrum reveals the presence of two stereoisomers, the one characterized crystallographically and a contribution of about 10% from a second symmetrical (sym) isomer wherein both dppv ligands occupy apical-basal sites. The unsymmetrical (unsym) arrangement of the dppv ligands is reflected in the values of A((31)P), which range from 31 MHz for the basal phosphines to 284 MHz for the apical phosphine. Density functional theory calculations were employed to rationalize the electronic structure of [H1](0) and to facilitate spectral simulation and assignment of EPR parameters including (1)H and (31)P hyperfine couplings. The EPR spectra of [H1](0) and [D1](0) demonstrate that the singly occupied molecular orbital is primarily localized on the Fe center with the longer bond to H, that is, Fe(II)-H···Fe(I). The coupling to the hydride is A((1)H) = 55 and 74 MHz for unsym- amd sym-[H1](0), respectively. Treatment of [H1](0) with H(+) gives 0.5 equiv of H2 and [H1](+). Reduction of D(+) affords D2, leaving the hydride ligand intact. These experiments demonstrate that the bridging hydride ligand in this complex is a spectator in the hydrogen evolution reaction.

Energy transducing redox steps of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

Na(+)-NQR is a unique respiratory enzyme that couples the free energy of electron transfer reactions to electrogenic pumping of sodium across the cell membrane. This enzyme is found in many marine and pathogenic bacteria where it plays an analogous role to the H(+)-pumping complex I. It has generally been assumed that the sodium pump of Na(+)-NQR operates on the basis of thermodynamic coupling between reduction of a single redox cofactor and the binding of sodium at a nearby site. In this study, we have defined the coupling to sodium translocation of individual steps in the redox reaction of Na(+)-NQR. Sodium uptake takes place in the reaction step in which an electron moves from the 2Fe-2S center to FMN(C), while the translocation of sodium across the membrane dielectric (and probably its release into the external medium) occurs when an electron moves from FMN(B) to riboflavin. This argues against a single-site coupling model because the redox steps that drive these two parts of the sodium pumping process do not have any redox cofactor in common. The significance of these results for the mechanism of coupling is discussed, and we proposed that Na(+)-NQR operates through a novel mechanism based on kinetic coupling, mediated by conformational changes.

Bioorganometallic mechanism of action, and inhibition, of IspH.

We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K ( M ),V (max)), EPR and (1)H, (2)H, (13)C, (31)P, and (57)Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on (57)Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a pi/sigma "metallacycle" or eta (2)-alkenyl complex. The complex is poised to interact with H(+) from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an eta (1)-allyl complex. After reduction, this forms an eta (3)-allyl pi-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by (1)H, (2)H, and (13)C ENDOR, where hyperfine couplings of approximately 6 MHz for (13)C and 10 MHz for (1)H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe(4)S(4)-containing proteins, such as IspG.

Mixed-valence nickel-iron dithiolate models of the [NiFe]-hydrogenase active site.

A series of mixed-valence nickel-iron dithiolates is described. Oxidation of (diphosphine)Ni(dithiolate)Fe(CO)(3) complexes 1, 2, and 3 with ferrocenium salts affords the corresponding tricarbonyl cations [(dppe)Ni(pdt)Fe(CO)(3)](+) ([1](+)), [(dppe)Ni(edt)Fe(CO)(3)](+) ([2](+)) and [(dcpe)Ni(pdt)Fe(CO)(3)](+) ([3](+)), respectively, where dppe = Ph(2)PCH(2)CH(2)PPh(2), dcpe = Cy(2)PCH(2)CH(2)PCy(2), (Cy = cyclohexyl), pdtH(2) = HSCH(2)CH(2)CH(2)SH, and edtH(2) = HSCH(2)CH(2)SH. The cation [2](+) proved unstable, but the propanedithiolates are robust. IR and EPR spectroscopic measurements indicate that these species exist as C(s)-symmetric species. Crystallographic characterization of [3]BF(4) shows that Ni is square planar. Interaction of [1]BF(4) with P-donor ligands (L) afforded a series of substituted derivatives of type [(dppe)Ni(pdt)Fe(CO)(2)L]BF(4) for L = P(OPh)(3) ([4a]BF(4)), P(p-C(6)H(4)Cl)(3) ([4b]BF(4)), PPh(2)(2-py) ([4c]BF(4)), PPh(2)(OEt) ([4d]BF(4)), PPh(3) ([4e]BF(4)), PPh(2)(o-C(6)H(4)OMe) ([4f]BF(4)), PPh(2)(o-C(6)H(4)OCH(2)OMe) ([4g]BF(4)), P(p-tol)(3) ([4h]BF(4)), P(p-C(6)H(4)OMe)(3) ([4i]BF(4)), and PMePh(2) ([4j]BF(4)). EPR analysis indicates that ethanedithiolate [2](+) exists as a single species at 110 K, whereas the propanedithiolate cations exist as a mixture of two conformers, which are proposed to be related through a flip of the chelate ring. Mössbauer spectra of 1 and oxidized S = 1/2 [4e]BF(4) are both consistent with a low-spin Fe(I) state. The hyperfine coupling tensor of [4e]BF(4) has a small isotropic component and significant anisotropy. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the SOMOs in complexes of the present type are localized in an Fe(I)-centered d(z(2)) orbital. The DFT calculations allow an assignment of oxidation states of the metals and rationalization of the conformers detected by EPR spectroscopy. Treatment of [1](+) with CN(-) and compact basic phosphines results in complex reactions. With dppe, [1](+) undergoes quasi-disproportionation to give 1 and the diamagnetic complex [(dppe)Ni(pdt)Fe(CO)(2)(dppe)](2+) ([5](2+)), which features square-planar Ni linked to an octahedral Fe center.