Kyle M. Lancaster

X-ray Emission Spectroscopy Evidences a Central Carbon in the Nitrogenase Iron-Molybdenum Cofactor

A central light atom in a cofactor at the nitrogenase active site is identified as a carbon. Nitrogenase is a complex enzyme that catalyzes the reduction of dinitrogen to ammonia. Despite insight from structural and biochemical studies, its structure and mechanism await full characterization. An iron-molybdenum cofactor (FeMoco) is thought to be the site of dinitrogen reduction, but the identity of a central atom in this cofactor remains unknown. Fe Kβ x-ray emission spectroscopy (XES) of intact nitrogenase MoFe protein, isolated FeMoco, and the FeMoco-deficient ∆nifB protein indicates that among the candidate atoms oxygen, nitrogen, and carbon, it is carbon that best fits the XES data. The experimental XES is supported by computational efforts, which show that oxidation and spin states do not affect the assignment of the central atom to C4–. Identification of the central atom will drive further studies on its role in catalysis.

Ultrafast excited-state dynamics of rhenium(I) photosensitizers [Re(Cl)(CO)3(N,N)] and [Re(imidazole)(CO)3(N,N)]+: diimine effects.

Femto- to picosecond excited-state dynamics of the complexes [Re(L)(CO)(3)(N,N)](n) (N,N = bpy, phen, 4,7-dimethyl-phen (dmp); L = Cl, n = 0; L = imidazole, n = 1+) were investigated using fluorescence up-conversion, transient absorption in the 650-285 nm range (using broad-band UV probe pulses around 300 nm) and picosecond time-resolved IR (TRIR) spectroscopy in the region of CO stretching vibrations. Optically populated singlet charge-transfer (CT) state(s) undergo femtosecond intersystem crossing to at least two hot triplet states with a rate that is faster in Cl (∼100 fs)(-1) than in imidazole (∼150 fs)(-1) complexes but essentially independent of the N,N ligand. TRIR spectra indicate the presence of two long-lived triplet states that are populated simultaneously and equilibrate in a few picoseconds. The minor state accounts for less than 20% of the relaxed excited population. UV-vis transient spectra were assigned using open-shell time-dependent density functional theory calculations on the lowest triplet CT state. Visible excited-state absorption originates mostly from mixed L;N,N(•-) → Re(II) ligand-to-metal CT transitions. Excited bpy complexes show the characteristic sharp near-UV band (Cl, 373 nm; imH, 365 nm) due to two predominantly ππ*(bpy(•-)) transitions. For phen and dmp, the UV excited-state absorption occurs at ∼305 nm, originating from a series of mixed ππ* and Re → CO;N,N(•-) MLCT transitions. UV-vis transient absorption features exhibit small intensity- and band-shape changes occurring with several lifetimes in the 1-5 ps range, while TRIR bands show small intensity changes (≤5 ps) and shifts (∼1 and 6-10 ps) to higher wavenumbers. These spectral changes are attributable to convoluted electronic and vibrational relaxation steps and equilibration between the two lowest triplets. Still slower changes (≥15 ps), manifested mostly by the excited-state UV band, probably involve local-solvent restructuring. Implications of the observed excited-state behavior for the development and use of Re-based sensitizers and probes are discussed.

Direct Spectroscopic Characterization of a Transitory Dirhodium Donor-Acceptor Carbene Complex

Catching a Carbene >> Divalent carbon fragments, or carbenes, vary widely in their stability, depending on their substituents. Some, such as N-heterocyclic carbenes, are independently isolatable. Others can be isolated in coordination complexes with metals. Kornecki et al. (p. 351, published online 12 September) synthesized a carbene coordinated to a rhodium dimer representative of an elusive class of short-lived intermediates long postulated to underlie a series of cyclopropanation and C-H insertion reactions. A long-postulated reactive chemical intermediate has been observed and characterized. A multitude of organic transformations catalyzed by dirhodium(II) (Rh2) complexes are thought to proceed via the intermediacy of highly reactive, electrophilic carbenoid intermediates that have eluded direct observation. Herein, we report the generation of a metastable Rh2-carbenoid intermediate supported by a donor-acceptor carbene fragment. This intermediate is stable for a period of ~20 hours in chloroform solution at 0°C, allowing for an exploration of its physical and chemical properties. The Rh=C bond, characterized by vibrational and nuclear magnetic resonance spectroscopy, extended x-ray absorption fine structure analysis, and quantum-chemical calculations, has weak σ and π components. This intermediate performs stoichiometric cyclopropanation and C–H functionalization reactions to give products that are identical to those obtained from analogous Rh2 catalysis.

Type-zero copper proteins

Copper proteins play key roles in biological processes such as electron transfer and dioxygen activation; the active site of each of these proteins is classified as either type 1, 2, or 3, depending on its optical and electron paramagnetic resonance properties. We have built a new type of site that we call “type zero copper” by incorporating leucine, isoleucine, or phenylalanine in place of methionine at position 121 in C112D Pseudomonas aeruginosa azurin. X-ray crystallographic analysis shows that these sites adopt distorted tetrahedral geometries, with an unusually short Cu-O(G45 carbonyl) bond (2.35–2.55 Å). Relatively weak absorption near 800 nm and narrow parallel hyperfine splittings in EPR spectra are the spectroscopic signatures of type zero copper. Copper K-edge x-ray absorption spectra suggest elevated Cu(II) 4p character in the d-electron ground state. Cyclic voltammetric experiments demonstrate that the electron transfer reactivities of type zero azurins are enhanced relative to that of the corresponding type 2 (C112D) protein.

Kβ X-ray emission spectroscopy offers unique chemical bonding insights: revisiting the electronic structure of ferrocene.

Kβ X-ray emission spectroscopy (XES) is emerging as a powerful tool for the study of chemical bonding. Analyses of the Kβ XES of ferrocene (Fc) and ferrocenium (Fc(+)) are presented as further demonstrations of the capabilities of the technique. Assignments of the valence to core (V2C) region of these spectra as electric dipole-allowed cyclopentadienyl (Cp) → Fe 1s transitions demonstrate that XES affords electronic structural insight into the energetics of ligand-based molecular orbitals (MOs). Combined with K-edge X-ray absorption spectroscopy (XAS), we show that XES can provide analogous information to photoemission spectroscopy (PES). Density functional theory (DFT) analyses reveal that the V2C transitions in Fc/Fc(+) derive their intensity from Fe 4p admixture (on the order of 5-10%) into the Cp-based MOs from which they originate. These 4p admixtures confer bonding character to the Cp-based a(2u) and e(1u) MOs to at least the extent of backbonding contributions to frontier MOs from higher-lying Cp π* MOs.

Inner- and outer-sphere metal coordination in blue copper proteins

Blue copper proteins (BCPs) comprise classic cases of Natures profound control over the electronic structures and chemical reactivity of transition metal ions. Early studies of BCPs focused on their inner coordination spheres, that is, residues that directly coordinate Cu. Equally important are the electronic and geometric perturbations to these ligands provided by the outer coordination sphere. In this tribute to Hans Freeman, we review investigations that have advanced the understanding of how inner-sphere and outer-sphere coordination affects biological Cu properties.

Electron Transfer Reactivity of Type Zero Pseudomonas aeruginosa Azurin

Type zero copper is a hard-ligand analogue of the classical type 1 or blue site in copper proteins that function as electron transfer (ET) agents in photosynthesis and other biological processes. The EPR spectroscopic features of type zero Cu(II) are very similar to those of blue copper, although lacking the deep blue color, due to the absence of thiolate ligation. We have measured the rates of intramolecular ET from the pulse radiolytically generated C3-C26 disulfide radical anion to the Cu(II) in both type zero C112D/M121L and type 2 C112D Pseudomonas aeruginosa azurins in pH 7.0 aqueous solutions between 8 and 45 °C. We also have obtained rate/temperature (10-30 °C) profiles for ET reactions between these mutants and the wild-type azurin. Analysis of the rates and activation parameters for both intramolecular and intermolecular ET reactions indicates that the type zero copper reorganization energy falls in a range (0.9-1.1 eV) slightly above that for type 1 (0.7-0.8 eV), but substantially smaller than that for type 2 (>2 eV), consistent with XAS and EXAFS data that reveal minimal type zero site reorientation during redox cycling.

Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase

Significance The enzymatic reactions that occur during nitrification are nature’s means to use ammonia as cellular fuel. Complete understanding of nitrification and related processes are vital to sustainable agriculture and renewable energy technologies. The prevailing view of the first phase of nitrification is that ammonia oxidizing bacteria use two enzymes, ammonia monooxygenase and hydroxylamine oxidoreductase, to oxidize ammonia to nitrite via hydroxylamine as an obligate intermediate. Our work reveals nitric oxide as an additional obligate intermediate. The presented findings necessitate revision of a key biogeochemical process, identify a new bioenergetic role for nitric oxide, predict participation of a third enzyme in the biological oxidation of ammonia to nitrite, and will inform models toward sustainable agriculture. Ammonia (NH3)-oxidizing bacteria (AOB) emit substantial amounts of nitric oxide (NO) and nitrous oxide (N2O), both of which contribute to the harmful environmental side effects of large-scale agriculture. The currently accepted model for AOB metabolism involves NH3 oxidation to nitrite (NO2–) via a single obligate intermediate, hydroxylamine (NH2OH). Within this model, the multiheme enzyme hydroxylamine oxidoreductase (HAO) catalyzes the four-electron oxidation of NH2OH to NO2–. We provide evidence that HAO oxidizes NH2OH by only three electrons to NO under both anaerobic and aerobic conditions. NO2– observed in HAO activity assays is a nonenzymatic product resulting from the oxidation of NO by O2 under aerobic conditions. Our present study implies that aerobic NH3 oxidation by AOB occurs via two obligate intermediates, NH2OH and NO, necessitating a mediator of the third enzymatic step.

Structures and Reactivity Patterns of Group 9 Metallocorroles

Group 9 metallocorroles 1-M(PPh(3)) and 1-M(py)(2) [M = Co(III), Rh(III), Ir(III); 1 denotes the trianion of 5,10,15-tris-pentafluorophenylcorrole] have been fully characterized by structural, spectroscopic, and electrochemical methods. Crystal structure analyses reveal that average metal-N(pyrrole) bond lengths of the bis-pyridine metal(III) complexes increase from Co (1.886 A) to Rh (1.957 A)/Ir (1.963 A); and the average metal-N(pyridine) bond lengths also increase from Co (1.995 A) to Rh (2.065 A)/Ir (2.059 A). Ligand affinities for 1-M(PPh(3)) axial coordination sites increase dramatically in the order 1-Co(PPh(3)) < 1-Rh(PPh(3)) < 1-Ir(PPh(3)). There is a surprising invariance in the M(+/0) reduction potentials within the five- and six-coordinate corrole series, and even between them; the average M(+/0) potential of 1-M(PPh(3)) is 0.78 V vs Ag/AgCl in CH(2)Cl(2) solution, whereas that of 1-M(py)(2) is 0.70 V under the same conditions. Electronic structures of one-electron-oxidized 1-M(py)(2) complexes have been assigned by analysis of electron paramagnetic resonance spectroscopic measurements: oxidation is corrole-centered for 1-Co(py)(2) (g = 2.008) and 1-Rh(py)(2) (g = 2.003), and metal-centered for 1-Ir(tma)(2) (g(zz) = 2.489, g(yy) = 2.010, g(xx) = 1.884, g(av) = 2.128) and 1-Ir(py)(2) (g(zz) = 2.401, g(yy) = 2.000, g(xx) = 1.937, g(av) = 2.113).

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission

Significance Nitrous oxide (N2O) is a potent ozone-depleting greenhouse gas. This work identifies a means by which N2O is generated during nitrification, or biological ammonia oxidation. Fertilizer use in agriculture stimulates nitrification, thus increasing the volume of N2O emissions worldwide. The results presented herein will inform models and strategies toward optimized, sustainable agriculture. Moreover, these results highlight a rare example of biological N–N bond formation. Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N2O). It has been proposed that N2O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH2OH) quantitatively to N2O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH2OH to nitrite (NO2−) under aerobic conditions. Although we observe NO2− formation under aerobic conditions, its concentration is not stoichiometric with the NH2OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH2OH to N2O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an FeIII–NH2OH adduct of cyt P460 is oxidized to an {FeNO}6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH2OH, forming the N–N bond of N2O during a bimolecular, rate-determining step. We propose that NO2− results when nitric oxide (NO) dissociates from the {FeNO}6 intermediate and reacts with dioxygen. Thus, NO2− is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH2OH contributes to NO and N2O emissions from nitrifying microorganisms.