Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Four Cu(I) complexes were synthesized with a family of pyridylmethylamide ligands, HLR [HLR = N-(2-pyridylmethyl)acetamide, R = null; 2,2-dimethyl-N-(2-pyridylmethyl)propionamide, R = Me3; 2,2,2-triphenyl-N-(2-pyridylmethyl)acetamide, R = Ph3)]. Complexes 1–3 were synthesized from the respective ligand and [Cu(CH3CN)4]PF6 in a 2 : 1 molar ratio: [Cu(HL)2]PF6 (1), [Cu2(HLMe3)4](PF6)2 (2), [Cu(HLPh3)2]PF6 (3). Complex 4, [Cu(HL)(CH3CN)(PPh3)]PF6, was synthesized from the reaction of HL with [Cu(CH3CN)4]PF6 and PPh3 in a 1 : 1 : 1 molar ratio. X-Ray crystal structures reveal that complexes 1, 3 and 4 are mononuclear Cu(I) species, while complex 2 is a Cu(I) dimer. The copper ions are four-coordinate with geometries ranging from distorted tetrahedral to seesaw in 1, 2, and 4. Complexes 1 and 2 are very air sensitive and they display similar electrochemical properties. The coordination geometry of complex 3 is nearly linear, two-coordinate. Complex 3 is exceptionally stable with respect to oxidation in the air, and its cyclic voltammetry shows no oxidation wave in the range of 0–1.5 V. The unusual inertness of complex 3 towards oxidation is attributed to the protection from bulky triphenyl substituent of the HLPh3 ligand. A new geometric parameter for four-coordinate compounds, τ4, is proposed as an improved, simple metric for quantitatively evaluating the geometry of four-coordinate complexes and compounds.

Structural variation in copper(I) complexes with pyridylmethylamide ligands: structural analysis with a new four-coordinate geometry index, tau4.

The purpose of this critical review is to give a representative and comprehensive overview of the arising developments in the field of magnetic metal–organic frameworks, in particular those containing cobalt(II). We examine the diversity of magnetic exchange interactions between nearest-neighbour moment carriers, covering from dimers to oligomers and discuss their implications in infinite chains, layers and networks, having a variety of topologies. We progress to the different forms of short-range magnetic ordering, giving rise to single-molecule-magnets and single-chain-magnets, to long-range ordering of two- and three-dimensional networks (323 references).

Magnetic metal-organic frameworks.

This critical review presents a comprehensive study of transition-metal carboxylate clusters which may serve as secondary building units (SBUs) towards construction and synthesis of metal–organic frameworks (MOFs). We describe the geometries of 131 SBUs, their connectivity and composition. This contribution presents a comprehensive list of the wide variety of transition-metal carboxylate clusters which may serve as secondary building units (SBUs) in the construction and synthesis of metal–organic frameworks. The SBUs discussed here were obtained from a search of molecules and extended structures archived in the Cambridge Structure Database (CSD, version 5.28, January 2007) which included only crystals containing metal carboxylate linkages (241 references).

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Secondary building units, nets and bonding in the chemistry of metal–organic frameworks

Based on its generally accessible I/II redox couple and bioavailability, copper plays a wide variety of roles in nature that mostly involve electron transfer (ET), O2 binding, activation and reduction, NO2− and N2O reduction and substrate activation. Copper sites that perform ET are the mononuclear blue Cu site that has a highly covalent CuII-S(Cys) bond and the binuclear CuA site that has a Cu2S(Cys)2 core with a Cu-Cu bond that keeps the site delocalized (Cu(1.5)2) in its oxidized state. In contrast to inorganic Cu complexes, these metalloprotein sites transfer electrons rapidly often over long distances, as has been previously reviewed.1–4 Blue Cu and CuA sites will only be considered here in their relation to intramolecular ET in multi-center enzymes. The focus of this review is on the Cu enzymes (Figure 1). Many are involved in O2 activation and reduction, which has mostly been thought to involve at least two electrons to overcome spin forbiddenness and the low potential of the one electron reduction to superoxide (Figure 2).5,6 Since the Cu(III) redox state has not been observed in biology, this requires either more than one Cu center or one copper and an additional redox active organic cofactor. The latter is formed in a biogenesis reaction of a residue (Tyr) that is also Cu catalyzed in the first turnover of the protein. Recently, however, there have been a number of enzymes suggested to utilize one Cu to activate O2 by 1e− reduction to form a Cu(II)-O2•− intermediate (an innersphere redox process) and it is important to understand the active site requirements to drive this reaction. The oxidases that catalyze the 4e−reduction of O2 to H2O are unique in that they effectively perform this reaction in one step indicating that the free energy barrier for the second two-electron reduction of the peroxide product of the first two-electron step is very low. In nature this requires either a trinuclear Cu cluster (in the multicopper oxidases) or a Cu/Tyr/Heme Fe cluster (in the cytochrome oxidases). The former accomplishes this with almost no overpotential maximizing its ability to oxidize substrates and its utility in biofuel cells, while the latter class of enzymes uses the excess energy to pump protons for ATP synthesis. In bacterial denitrification, a mononuclear Cu center catalyzes the 1e- reduction of nitrite to NO while a unique µ4S2−Cu4 cluster catalyzes the reduction of N2O to N2 and H2O, a 2e− process yet requiring 4Cu’s. Finally there are now several classes of enzymes that utilize an oxidized Cu(II) center to activate a covalently bound substrate to react with O2.



Figure 1

Copper active sites in biology.





Figure 2

Latimer Diagram for Oxygen Reduction at pH = 7.0 Adapted from References 5 and 6.



This review presents in depth discussions of all these classes of Cu enzymes and the correlations within and among these classes. For each class we review our present understanding of the enzymology, kinetics, geometric structures, electronic structures and the reaction mechanisms these have elucidated. While the emphasis here is on the enzymology, model studies have significantly contributed to our understanding of O2 activation by a number of Cu enzymes and are included in appropriate subsections of this review. In general we will consider how the covalency of a Cu(II)–substrate bond can activate the substrate for its spin forbidden reaction with O2, how in binuclear Cu enzymes the exchange coupling between Cu’s overcomes the spin forbiddenness of O2 binding and controls electron transfer to O2 to direct catalysis either to perform two e− electrophilic aromatic substitution or 1e− H-atom abstraction, the type of oxygen intermediate that is required for H-atom abstraction from the strong C-H bond of methane (104 kcal/mol) and how the trinuclear Cu cluster and the Cu/Tyr/Heme Fe cluster achieve their very low barriers for the reductive cleavage of the O-O bond.

Much of the insight available into these mechanisms in Cu biochemistry has come from the application of a wide range of spectroscopies and the correlation of spectroscopic results to electronic structure calculations. Thus we start with a tutorial on the different spectroscopic methods utilized to study mononuclear and multinuclear Cu enzymes and their correlations to different levels of electronic structure calculations.

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Copper Active Sites in Biology

A thiolate-bridged, fully delocalized mixed-valence dicopper(I,II) complex that models the CuA biological electron-transfer site

Using a combination of electronic spectroscopies, electronic structural descriptions have been developed for a series of binuclear CuA-type centers in Bacillus subtilis CcO and engineered into the blue copper proteins Pseudomonas aeruginosa azurin and Thiobacillus versutus amicyanin. Parallel descriptions are developed for two structurally characterized mixed-valence (MV) and homovalent (II,II) synthetic copper thiolate dimers. Assignment of the excited-state spectral features allows the electronic structures of CuA and the MV model to be understood and compared in relation to their copper coordination environments. These electronic structural descriptions are supported by SCF-Xα-SW MO calculations, which are used to test systematically the effects of major structural perturbations linking the MV model geometry to that of CuA. It is determined that both Cu−Cu compression and removal of the axial ligands are critical determinants of the orbital ground state in these dimers. The weakened axial interactions ...

Spectroscopy of mixed-valence Cu(A)-type centers: Ligand-field control of ground-state properties related to electron transfer

The coordination polymers {[Cu3(TMA)2(Im)6(H2O)]·7H2O}n (1) and {[Cu3(TMA)2(Me-Im)6(H2O)]·16H2O}n (2) were synthesized by combining copper sulfate, TMA, and either Im or Me-Im in water. Single-crystal X-ray diffraction studies revealed that both compounds are composed of 2D sheets of Cu2+ cations and TMA3- anions. Hydrogen bonding between sheets in 1 stabilizes its 3D structure, while 2 is destabilized because of the absence of intersheet hydrogen bonding.

Coordination polymers composed of copper(II), trimesic acid, and imidazole: 3D architecture stabilized by hydrogen bonding.

Cytochrome oxidase is the terminal oxidase in both prokaryotic and eukaryotic cells and is responsible for the generation of cellular energy via the process known as oxidative phosphorylation. The enzyme contains two Fe and three Cu centers which together provide the redox machinery for the reduction of O2 to water. Recently, X-ray crystallography has provided the first three-dimensional description of the coordination spheres of the metal centers. However, the structures show the metal sites at low resolution, and in order to fully understand the mechanism of the reaction, it is desirable to determine the metrical details (bond lengths and angles) to much higher precision. X-ray absorption spectroscopy is unique in its ability to provide such detail, and we have applied the technique to determining the structure of the CuA center, a thiolate-bridged binuclear copper cluster in which the coppers are bridged by two cysteine ligands and have an extremely short Cu−Cu distance of ∼2.4 A. X-ray absorption spec...

Robert P. Houser

Papers

Structural variation in copper(I) complexes with pyridylmethylamide ligands: structural analysis with a new four-coordinate geometry index, tau4.

A thiolate-bridged, fully delocalized mixed-valence dicopper(I,II) complex that models the CuA biological electron-transfer site

Spectroscopy of mixed-valence Cu(A)-type centers: Ligand-field control of ground-state properties related to electron transfer

Coordination polymers composed of copper(II), trimesic acid, and imidazole: 3D architecture stabilized by hydrogen bonding.

X-ray Absorption Studies on the Mixed-Valence and Fully Reduced Forms of the Soluble CuA Domains of Cytochrome c Oxidase