Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster.

Nitrogenase catalyzes the multielectron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxidation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, ßSer188, was mutated to a noncoordinating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av ßSer188Ala MoFeP) displayed the same diazotrophic growth and in vitro enzyme turnover rates as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that ßSer188 plays a role in protecting the P-cluster under nonideal conditions. Here, we investigated the protective role of ßSer188 both in vivo and in vitro by characterizing the ability of Av ßSer188Ala cells to grow under suboptimal conditions (high oxidative stress or Fe limitation) and by determining the tendency of ßSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that ßSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the expanding functional roles of non-Cys ligands to FeS clusters.

17O Electron Nuclear Double Resonance Analysis of Compound I: Inverse Correlation between Oxygen Spin Population and Electron Donation.

Although the activation of inert C-H bonds by metal-oxo complexes has been widely studied, important questions remain, particularly regarding the role of oxygen spin population (i.e., unpaired electrons on the oxo ligand) in facilitating C-H bond cleavage. In order to shed light on this issue, we have utilized 17O electron nuclear double resonance spectroscopy to measure the oxygen spin populations of three compound I intermediates in heme enzymes with different reactivities toward C-H bonds: chloroperoxidase, cytochrome P450, and a selenolate (selenocysteinyl)-ligated cytochrome P450. The experimental data suggest an inverse correlation between oxygen spin population and electron donation from the axial ligand. We have explored the implications of this result using a Hückel-type molecular orbital model and constrained density functional theory calculations. These investigations have allowed us to examine the relationship between oxygen spin population, oxygen charge, electron donation from the axial ligand, and reactivity.

NRVS investigation of ascorbate peroxidase compound II: Observation of Iron(IV)oxo stretching.

The protonation state of ascorbate peroxidase compound II (APX-II) has been a subject of debate. A combined X-ray/neutron crystallographic study reported that APX-II is best described as an iron(IV)hydroxide species with an FeO distance of 1.88 Å (Kwon, et al. Nat Commun2016, 7, 13,445), while X-ray absorption spectroscopy (XAS) experiments (utilizing extended X-ray absorption fine structure (EXAFS) and pre-edge analyses) indicate APX-II is an authentic iron(IV)oxo species with an FeO distance 1.68 Å (Ledray, et al. Journal of the American Chemical Society2020,142, 20,419). Previous debates concerning ferryl protonation states have been resolved through the application of Badger's rule, which correlates FeO bond distances with FeO vibrational frequencies. To obtain the required vibrational data, we have collected Nuclear Resonance Vibrational Spectroscopy (NRVS) data for APX-II. We observe a broad vibrational feature in the range associated with iron(IV)oxo stretching (700-800 cm-1). This feature appears to have two peaks at 732 cm-1 and 770 cm-1, corresponding to FeO distances of 1.69 and 1.67 Å, respectively. The broad vibrational envelope and the presence of multiple resonances could reflect a distribution of hydrogen bonding interactions within the active-site pocket.

Semiempirical method for examining asynchronicity in metal-oxido-mediated C-H bond activation.

The oxidation of substrates via the cleavage of thermodynamically strong C-H bonds is an essential part of mammalian metabolism. These reactions are predominantly carried out by enzymes that produce high-valent metal-oxido species, which are directly responsible for cleaving the C-H bonds. While much is known about the identity of these transient intermediates, the mechanistic factors that enable metal-oxido species to accomplish such difficult reactions are still incomplete. For synthetic metal-oxido species, C-H bond cleavage is often mechanistically described as synchronous, proton-coupled electron transfer (PCET). However, data have emerged that suggest that the basicity of the M-oxido unit is the key determinant in achieving enzymatic function, thus requiring alternative mechanisms whereby proton transfer (PT) has a more dominant role than electron transfer (ET). To bridge this knowledge gap, the reactivity of a monomeric MnIV-oxido complex with a series of external substrates was studied, resulting in a spread of over 104 in their second-order rate constants that tracked with the acidity of the C-H bonds. Mechanisms that included either synchronous PCET or rate-limiting PT, followed by ET, did not explain our results, which led to a proposed PCET mechanism with asynchronous transition states that are dominated by PT. To support this premise, we report a semiempirical free energy analysis that can predict the relative contributions of PT and ET for a given set of substrates. These findings underscore why the basicity of M-oxido units needs to be considered in C-H functionalization.

Diazophosphonates: Effective Surrogates for Diazoalkanes in Pyrazole Synthesis.

Diazophosphonates, readily prepared from α-ketophosphonates by oxidation of the corresponding hydrazones in batch or in flow, are useful partners in 1,3-dipolar cycloaddition reactions to alkynes to give N-H pyrazoles, including the first intramolecular examples of such a process. The phosphoryl group imbues a number of desirable properties into the diazo 1,3-dipole. The electron-withdrawing nature of the phosphoryl stabilizes the diazo compound making it easier to handle, whilst the ability of the phosphoryl group to migrate readily in a [1,5]-sigmatropic rearrangement enables its transfer from C to N to aromatize the initial cycloadduct, and hence its facile removal from the final pyrazole product. Overall, the diazophosphonate acts as a surrogate for the much less stable diazoalkane in cycloadditions, with the phosphoryl group playing a vital, but traceless, role. The cycloaddition proceeds more readily with alkynes bearing electron-withdrawing groups, and is regiospecific with asymmetrical alkynes. The potential of diazophosphonates for use in bioorthogonal cycloadditions is demonstrated by their facile addition to strained alkynes.

Different Kinetic Reactivity of Electrons in Distinct TiO2 Nanoparticle Trap States.

Electrons added to TiO2 and other semiconductors often occupy trap states, whose reactivity can determine the catalytic and stoichiometric chemistry of the material. We previously showed that reduced aqueous colloidal TiO2 nanoparticles have two distinct classes of thermally-equilibrated trapped electrons, termed Red/e - and Blue/e -. Presented here are parallel optical and electron paramagnetic resonance (EPR) kinetic studies of the reactivity of these electrons with solution-based oxidants. Optical stopped-flow measurements monitoring reactions of TiO2/e - with sub-stoichiometric oxidants showed a surprising pattern: an initial fast (seconds) decrease in TiO2/e - absorbance followed by a secondary, slow (minutes) increase in the broad TiO2/e - optical feature. Analysis revealed that the fast decrease is due to the preferential oxidation of the Red/e - trap states, and the slow increase results from re-equilibration of electrons from Blue to Red states. This kinetic model was confirmed by freeze-quench EPR measurements. Quantitative analysis of the kinetic data demonstrated that Red/e - react ~5 times faster than Blue/e - with the nitroxyl radical oxidant, 4-MeO-TEMPO. Similar reactivity patterns were also observed in oxidations of TiO2/e - by O2, which like 4-MeO-TEMPO is a proton-coupled electron transfer (PCET) oxidant, and by the pure electron transfer (ET) oxidant KI3. This suggests that the faster intrinsic reactivity of one trap state over another on the seconds-minutes timescale is likely a general feature of reduced TiO2 reactivity. This differential trap state reactivity is likely to influence the performance of TiO2 in photochemical/electrochemical devices, and it suggests an opportunity for tuning catalysis.

Ascorbate Peroxidase Compound II Is an Iron(IV) Oxo Species.

The protonation state of the iron(IV) oxo (or ferryl) form of ascorbate peroxidase compound II (APX-II) is a subject of debate. It has been reported that this intermediate is best described as an iron(IV) hydroxide species. Neutron diffraction data obtained from putative APX-II crystals indicate a protonated oxygenic ligand at 1.88 Å from the heme iron. This finding, if correct, would be unprecedented. A basic iron(IV) oxo species has yet to be spectroscopically observed in a histidine-ligated heme enzyme. The importance of ferryl basicity lies in its connection to our fundamental understanding of C-H bond activation. Basic ferryl species have been proposed to facilitate the oxidation of inert C-H bonds, reactions that are unknown for histidine-ligated hemes enzymes. To provide further insight into the protonation status of APX-II, we examined the intermediate using a combination of Mössbauer and X-ray absorption spectroscopies. Our data indicate that APX-II is an iron(IV) oxo species with an Fe-O bond distance of 1.68 Å, a K-edge pre-edge absorption of 18 units, and Mössbauer parameters of ΔEq = 1.65 mm/s and δ = 0.03 mm/s.

Effects of Noncovalent Interactions on High-Spin Fe(IV)-Oxido Complexes.

High-valent nonheme FeIV-oxido species are key intermediates in biological oxidation, and their properties are proposed to be influenced by the unique microenvironments present in protein active sites. Microenvironments are regulated by noncovalent interactions, such as hydrogen bonds (H-bonds) and electrostatic interactions; however, there is little quantitative information about how these interactions affect crucial properties of high valent metal-oxido complexes. To address this knowledge gap, we introduced a series of FeIV-oxido complexes that have the same S = 2 spin ground state as those found in nature and then systematically probed the effects of noncovalent interactions on their electronic, structural, and vibrational properties. The key design feature that provides access to these complexes is the new tripodal ligand [poat]3-, which contains phosphinic amido groups. An important structural aspect of [FeIVpoat(O)]- is the inclusion of an auxiliary site capable of binding a Lewis acid (LAII); we used this unique feature to further modulate the electrostatic environment around the Fe-oxido unit. Experimentally, studies confirmed that H-bonds and LAII s can interact directly with the oxido ligand in FeIV-oxido complexes, which weakens the Fe═O bond and has an impact on the electronic structure. We found that relatively large vibrational changes in the Fe-oxido unit correlate with small structural changes that could be difficult to measure, especially within a protein active site. Our work demonstrates the important role of noncovalent interactions on the properties of metal complexes, and that these interactions need to be considered when developing effective oxidants.

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases.

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.

Reduction Potentials of P450 Compounds I and II: Insight into the Thermodynamics of C-H Bond Activation.

We present a mixed experimental/theoretical determination of the bond strengths and redox potentials that define the ground-state thermodynamics for C-H bond activation in cytochrome P450 catalysis. Using redox titrations with [Ir(IV)Cl6]2-, we have determined the compound II/ferric (or Fe(IV)OH/Fe(III)OH2) couple and its associated D(O-H)Ferric bond strength in CYP158. Knowledge of this potential as well as the compound II/ferric (or Fe(IV)O/Fe(III)OH) reduction potential in horseradish peroxidase and the two-electron compound I/ferric (or Fe(IV)O(Por•)/Fe(III)OH2(Por)) reduction potential in aromatic peroxidase has allowed us to gauge the accuracy of theoretically determined bond strengths. Using the restricted open shell (ROS) method as proposed by Wright and co-workers, we have obtained O-H bond strengths and associated redox potentials for charge-neutral H-atom reductions of these iron(IV)-hydroxo and -oxo porphyrin species that are within 1 kcal/mol of experimentally determined values, suggesting that the ROS method may provide accurate values for the P450-II O-H bond strength and P450-I reduction potential. The efforts detailed here indicate that the ground-state thermodynamics of C-H bond activation in P450 are best described as follows: E0'Comp-I = 1.22 V (at pH 7, vs NHE) with D(O-H)Comp-II = 95 kcal/mol and E0'Comp-II = 0.99 V (at pH 7, vs NHE) with D(O-H)Ferric = 90 kcal/mol.

An efficient, step-economical strategy for the design of functional metalloproteins.

The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. Although numerous strategies have been used to create new metalloproteins, pre-existing knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (metal active sites by covalent tethering or MASCoT) in which folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naive protein-protein interfaces. Metalloproteins generated using this strategy uniformly bind a wide array of first-row transition metal ions (MnII, FeII, CoII, NiII, CuII, ZnII and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for MnII to 50 fM for CuII). MASCoT readily affords coordinatively unsaturated metal centres-including a penta-His-coordinated non-haem Fe site-and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands such as nitric oxide to the interfacial metal centre.

Nitrogen Fixation via a Terminal Fe(IV) Nitride.

Terminal iron nitrides (Fe≡N) have been proposed as intermediates of (bio)catalytic nitrogen fixation, yet experimental evidence to support this hypothesis has been lacking. In particular, no prior synthetic examples of terminal Fe≡N species have been derived from N2. Here we show that a nitrogen-fixing Fe-N2 catalyst can be protonated to form a neutral Fe(NNH2) hydrazido(2-) intermediate, which, upon further protonation, heterolytically cleaves the N-N bond to release [FeIV≡N]+ and NH3. These observations provide direct evidence for the viability of a Chatt-type (distal) mechanism for Fe-mediated N2-to-NH3 conversion. The physical oxidation state range of the Fe complexes in this transformation is buffered by covalency with the ligand, a feature of possible relevance to catalyst design in synthetic and natural systems that facilitate multiproton/multielectron redox processes.

Direct Observation of Oxygen Rebound with an Iron-Hydroxide Complex.

The rebound mechanism for alkane hydroxylation was invoked over 40 years ago to help explain reactivity patterns in cytochrome P450, and subsequently has been used to provide insight into a range of biological and synthetic systems. Efforts to model the rebound reaction in a synthetic system have been unsuccessful, in part because of the challenge in preparing a suitable metal-hydroxide complex at the correct oxidation level. Herein we report the synthesis of such a complex. The reaction of this species with a series of substituted radicals allows for the direct interrogation of the rebound process, providing insight into this uniformly invoked, but previously unobserved process.

Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity.

Strong electron-donation from the axial thiolate ligand of cytochrome P450 has been proposed to increase the reactivity of compound I with respect to C-H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we have prepared a selenolate-ligated cytochrome P450 compound I intermediate. This isoelectronic perturbation allows for direct comparisons with the wild-type enzyme. Selenium incorporation was achieved using a cysteine auxotrophic Escherichia coli strain. The intermediate was prepared with meta-chloroperbenzoic acid and characterized by UV-visible, Mössbauer and electron paramagnetic resonance spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate ligand. In line with this observation, we find that the selenolate-ligated compound I cleaves C-H bonds more rapidly than the wild-type intermediate.

A new look at the role of thiolate ligation in cytochrome P450.

Protonated ferryl (or iron(IV)hydroxide) intermediates have been characterized in several thiolate-ligated heme proteins that are known to catalyze C-H bond activation. The basicity of the ferryl intermediates in these species has been proposed to play a critical role in facilitating this chemistry, allowing hydrogen abstraction at reduction potentials below those that would otherwise lead to oxidative degradation of the enzyme. In this contribution, we discuss the events that led to the assignment and characterization of the unusual iron(IV)hydroxide species, highlighting experiments that provided a quantitative measure of the ferryl basicity, the iron(IV)hydroxide pKa. We then turn to the importance of the iron(IV)hydroxide state, presenting a new way of looking at the role of thiolate ligation in these systems.

Spectroscopic Investigations of Catalase Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non-thiolate-Ligated Heme Enzyme.

We report on the protonation state of Helicobacter pylori catalase compound II. UV/visible, Mössbauer, and X-ray absorption spectroscopies have been used to examine the intermediate from pH 5 to 14. We have determined that HPC-II exists in an iron(IV) hydroxide state up to pH 11. Above this pH, the iron(IV) hydroxide complex transitions to a new species (pKa = 13.1) with Mössbauer parameters that are indicative of an iron(IV)-oxo intermediate. Recently, we discussed a role for an elevated compound II pKa in diminishing the compound I reduction potential. This has the effect of shifting the thermodynamic landscape toward the two-electron chemistry that is critical for catalase function. In catalase, a diminished potential would increase the selectivity for peroxide disproportionation over off-pathway one-electron chemistry, reducing the buildup of the inactive compound II state and reducing the need for energetically expensive electron donor molecules.

Reactivity of an FeIV-Oxo Complex with Protons and Oxidants.

High-valent Fe-OH species are often invoked as key intermediates but have only been observed in Compound II of cytochrome P450s. To further address the properties of non-heme FeIV-OH complexes, we demonstrate the reversible protonation of a synthetic FeIV-oxo species containing a tris-urea tripodal ligand. The same protonated FeIV-oxo species can be prepared via oxidation, suggesting that a putative FeV-oxo species was initially generated. Computational, Mössbauer, XAS, and NRVS studies indicate that protonation of the FeIV-oxo complex most likely occurs on the tripodal ligand, which undergoes a structural change that results in the formation of a new intramolecular H-bond with the oxido ligand that aids in stabilizing the protonated adduct. We suggest that similar protonated high-valent Fe-oxo species may occur in the active sites of proteins. This finding further argues for caution when assigning unverified high-valent Fe-OH species to mechanisms.

Significantly shorter Fe-S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase.

Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I using variable-temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in the hydrogen-bonding patterns within the 'cys-pocket' (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation.

Experimental Correlation of Substrate Position with Reaction Outcome in the Aliphatic Halogenase, SyrB2.

The iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations, but how individual members of the enzyme family direct different outcomes is poorly understood. The Fe/2OG halogenase, SyrB2, chlorinates C4 of its native substrate, l-threonine appended to the carrier protein, SyrB1, but hydroxylates C5 of l-norvaline and, to a lesser extent, C4 of l-aminobutyric acid when SyrB1 presents these non-native amino acids. To test the hypothesis that positioning of the targeted carbon dictates the outcome, we defined the positions of these three substrates by measuring hyperfine couplings between substrate deuterium atoms and the stable, EPR-active iron-nitrosyl adduct, a surrogate for reaction intermediates. The Fe-(2)H distances and N-Fe-(2)H angles, which vary from 4.2 Å and 85° for threonine to 3.4 Å and 65° for norvaline, rationalize the trends in reactivity. This experimental correlation of position to outcome should aid in judging from structural data on other Fe/2OG enzymes whether they suppress hydroxylation or form hydroxylated intermediates on the pathways to other outcomes.

Oxygen-atom transfer reactivity of axially ligated Mn(V)-oxo complexes: evidence for enhanced electrophilic and nucleophilic pathways.

Addition of anionic donors to the manganese(V)-oxo corrolazine complex Mn(V)(O)(TBP8Cz) has a dramatic influence on oxygen-atom transfer (OAT) reactivity with thioether substrates. The six-coordinate anionic [Mn(V)(O)(TBP8Cz)(X)](-) complexes (X = F(-), N3(-), OCN(-)) exhibit a ∼5 cm(-1) downshift of the Mn-O vibrational mode relative to the parent Mn(V)(O)(TBP8Cz) complex as seen by resonance Raman spectroscopy. Product analysis shows that the oxidation of thioether substrates gives sulfoxide product, consistent with single OAT. A wide range of OAT reactivity is seen for the different axial ligands, with the following trend determined from a comparison of their second-order rate constants for sulfoxidation: five-coordinate ≈ thiocyanate ≈ nitrate < cyanate < azide < fluoride ≪ cyanide. This trend correlates with DFT calculations on the binding of the axial donors to the parent Mn(V)(O)(TBP8Cz) complex. A Hammett study was performed with p-X-C6H4SCH3 derivatives and [Mn(V)(O)(TBP8Cz)(X)](-) (X = CN(-) or F(-)) as the oxidant, and unusual "V-shaped" Hammett plots were obtained. These results are rationalized based upon a change in mechanism that hinges on the ability of the [Mn(V)(O)(TBP8Cz)(X)](-) complexes to function as either an electrophilic or weak nucleophilic oxidant depending upon the nature of the para-X substituents. For comparison, the one-electron-oxidized cationic Mn(V)(O)(TBP8Cz(•+)) complex yielded a linear Hammett relationship for all substrates (ρ = -1.40), consistent with a straightforward electrophilic mechanism. This study provides new, fundamental insights regarding the influence of axial donors on high-valent Mn(V)(O) porphyrinoid complexes.