Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium beijerinkii.

[FeFe] hydrogenase from Clostridium beijerinkii (CbHydA1) is an unusual hydrogenase in that it can withstand prolonged exposure to O2 by reversibly converting into an O2-protected, inactive state (Hinact). It has been indicated in the past that an atypical conformation of the "SC367CP" loop near the [2Fe]H portion of the six-iron active site (H-cluster) allows the Cys367 residue to adopt an "off-H+-pathway" orientation, promoting a facile transition of the cofactor to Hinact. Here, we investigated the electronic structure of the H-cluster in the oxidized state (Hox) that directly converts to Hinact under oxidizing conditions and the related CO-inhibited state (Hox-CO). We demonstrate that both states exhibit two distinct forms in electron paramagnetic resonance (EPR) spectroscopy. The ratio between the two forms is pH-dependent but also sensitive to the buffer choice. Our IR and EPR analyses illustrate that the spectral heterogeneity is due to a perturbation of the coordination environment of the H-cluster's [4Fe4S]H subcluster without affecting the [2Fe]H subcluster. Overall, we conclude that the observation of two spectral components per state is evidence of heterogeneity of the environment of the H-cluster likely associated with conformational mobility of the SCCP loop. Such flexibility may allow Cys367 to switch rapidly between off- and on-H+-pathway rotamers. Consequently, we believe such structural mobility may be the key to maintaining high enzymatic activity while allowing a facile transition to the O2-protected state.

Cytochrome P460 Cofactor Maturation Proceeds via Peroxide-Dependent Post-translational Modification.

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide. They bear specialized "heme P460" cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link-deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, this proenzyme undergoes maturation to active enzyme with spectroscopic and catalytic properties that match wild-type cyt P460. This maturation reactivity requires no chaperones─it is intrinsic to the protein. This behavior extends to the broader cytochrome c'ß superfamily. Accumulated data reveal key contributions from the secondary coordination sphere that enable selective, complete maturation. Spectroscopic data support the intermediacy of a ferryl species along the maturation pathway.

Biological and Bioinspired Inorganic N-N Bond-Forming Reactions.

The metallobiochemistry underlying the formation of the inorganic N-N-bond-containing molecules nitrous oxide (N2O), dinitrogen (N2), and hydrazine (N2H4) is essential to the lifestyles of diverse organisms. Similar reactions hold promise as means to use N-based fuels as alternative carbon-free energy sources. This review discusses research efforts to understand the mechanisms underlying biological N-N bond formation in primary metabolism and how the associated reactions are tied to energy transduction and organismal survival. These efforts comprise studies of both natural and engineered metalloenzymes as well as synthetic model complexes.

Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2.

All radical S-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1-Dph2, require at least one [4Fe-4S](Cys)3 cluster for activity. It is well-known in the radical-SAM enzyme community that the [4Fe-4S](Cys)3 cluster is extremely air-sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1-Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe-4S] cluster is easily degraded into a [3Fe-4S] cluster. Remarkably, the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. This mechanism to maintain radical-SAM enzyme activity in aerobic environments is likely general, and Dph3-like proteins may exist to keep other radical-SAM enzymes functional in aerobic environments.

An Approach to Carbide-Centered Cluster Complexes.

We report the first examples of the carbide ligand in (Cy3P)2Cl2Ru≡C (RuC) developing into a µ3 ligand toward metal centers. Conventionally, sterics exclude this coordination mode, but Fe2(CO)9 and Co2(CO)8 expel bridging CO ligands upon reaction with RuC to form trimetallic (Cy3P)2Cl2Ru═CFe2(CO)8 (RuCFe2) and (Cy3P)2Cl2Ru═CCo2(CO)7 (RuCCo2) complexes. Thus, the proximity offered by metal-metal associations in bimetallic carbonyl complexes allows the formation of trinuclear carbide complexes as verified by NMR, Mössbauer, and X-ray spectroscopic techniques.

4-Chloro-3-ethyl-phenol.

The title compound, C8H9ClO, packs with two independent mol-ecules in the asymmetric unit, without significant differences in corresponding bond lengths and angles, with the ethyl group in each oriented nearly perpendicular to the aromatic ring having ring-to-side chain torsion angles of 81.14 (18) and -81.06 (19)°. In the crystal, mol-ecules form an O-H⋯O hydrogen-bonded chain extending along the b-axis direction, through the phenol groups in which the H atoms are disordered. These chains pack together in the solid state, giving a sheet lying parallel to (001), via an offset face-to-face π-stacking inter-action characterized by a centroid-centroid distance of 3.580 (1) Å, together with a short inter-molecular Cl⋯Cl contact [3.412 (1) Å].

(E)-Benz-yl(1-phenyl-ethyl-idene)amine.

The title compound, C15H15N, represents an E isomer. The mol-ecule exhibits a minor [9.1 (2)%] disorder with methyl-benzyl-idene and benzyl groups inter-changing their positions. The C=N bond length is 1.292 (2) Å. The mol-ecular geometry is essentially planar, with the maximal twist of 8.5 (3)° for the benzyl group. The herringbone packing arrangement does not exhibit any π-stacking inter-actions.

Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460.

Product selectivity of ammonia oxidation by ammonia-oxidizing bacteria (AOB) is tightly controlled by metalloenzymes. Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO). The non-metabolic enzyme cytochrome (cyt) P460 also oxidizes NH2OH, but instead produces nitrous oxide (N2O). While both enzymes use a heme P460 cofactor, they selectively oxidize NH2OH to different products. Previously reported structures of Nitrosomonas sp. AL212 cyt P460 show that a capping phenylalanine residue rotates upon ligand binding, suggesting that this Phe may influence substrate and/or product binding. Here, we show via substitutions of the capping Phe in Nitrosomonas europaea cyt P460 that the bulky phenyl side-chain promotes the heme-lysine cross-link forming reaction operative in maturing the cofactor. Additionally, the Phe side-chain plays an important role in modulating product selectivity between N2O and NO during NH2OH oxidation under aerobic conditions. A picture emerges where the sterics and electrostatics of the side-chain in this capping position control the kinetics of N2O formation and NO binding affinity. This demonstrates how the outer coordination sphere of cyt P460 is tuned not only for selective NH2OH oxidation, but also for the autocatalytic cross-link forming reaction that imbues activity to an otherwise inactive protein.

Controlling a burn: outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460.

Ammonia oxidizing bacteria (AOB) use the cytotoxic, energetic molecule hydroxylamine (NH2OH) as a source of reducing equivalents for cellular respiration. Despite disproportionation or violent decomposition being typical outcomes of reactions of NH2OH with iron, AOB and anammox heme P460 proteins including cytochrome (cyt) P460 and hydroxylamine oxidoreductase (HAO) effect controlled, stepwise oxidation of NH2OH to nitric oxide (NO). Curiously, a recently characterized cyt P460 variant from the AOB Nitrosomonas sp. AL212 is able to form all intermediates of cyt P460 catalysis, but is nevertheless incompetent for NH2OH oxidation. We now show via site-directed mutagenesis, activity assays, spectroscopy, and structural biology that this lack of activity is attributable to the absence of a critical basic glutamate residue in the distal pocket above the heme P460 cofactor. This substitution is the only distinguishing characteristic of a protein that is otherwise effectively structurally and spectroscopically identical to an active variant. This highlights and reinforces a fundamental principal of metalloenzymology: metallocofactor inner-sphere geometric and electronic structures are in many cases insufficient for imbuing reactivity; a precisely defined outer coordination sphere contributed by the polypeptide matrix can be the key differentiator between a metalloenzyme and an unreactive metalloprotein.

Synthesis and crystal structure of a novel prochiral ketoimine: (E)-acetophenone O-diphenylphosphoryl oxime.

A novel activated prochiral ketoimine, (E)-acetophenone O-diphenylphosphoryl oxime, C(20)H(18)NO(2)P, with an electron-withdrawing substituent on the imine N atom similar to other prochiral ketoimines, has been synthesized and the X-ray crystal stucture determined. The molecules pack together in the solid state via weak intermolecular C-H...O interactions and both face-to-face and edge-to-face π-stacking interactions.