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.

/pdf/copper-active-sites-in-biology-lhhabv47if.pdf

Copper Active Sites in Biology

Department of Chemistry, The Johns Hopkins University, Charles and 34th Streets, Baltimore, Maryland 21218, Kluyver Laboratory for Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands, Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering at OHSU, Beaverton, Oregon 97006, and Department of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095

Nitric Oxide in Biological Denitrification: Fe/Cu Metalloenzyme and Metal Complex NOx Redox Chemistry

1. Overview A 2. Protein Redesign B 2.1. Making Use of Native Proteins: Protein Redesign B 2.2. Protein Redesign Based on Functions C 2.2.1. Redesign of Zinc Finger Structural Sites C 2.2.2. Redesign of Zinc Hydrolytic Centers E 2.2.3. Redesign of Heme Centers J 2.2.4. Redesign of Nonheme Redox Centers M 2.2.5. Artificial Metalloenzymes for Regioand Enantioselective Catalysis AA 2.2.6. Redesigned Protein Assemblies as Nanoreactors AK 2.3. Summary AN 3. De Novo Design AN 3.1. A Minimalist Approach: Designing Proteins from Scratch AN 3.2. Interactions between De Novo Designed Peptides and Metal Ions AN 3.2.1. Heavy Metal Toxicity AO 3.2.2. De Novo Designed Metal Centers Based on β-Structures AT 3.2.3. Metal-Induced Protein Folding AV 3.3. De Novo Designed Functional Metalloproteins: The Grail Quest of Protein Design AX 3.3.1. De Novo Designed Hydrolytic Centers AX 3.3.2. De Novo Designed Electron Transfer Centers BB 3.3.3. Other Catalytic Centers BG 3.4. Summary BN 4. Perspective BO Author Information BO Corresponding Author BO Notes BO Biographies BO Abbreviations BR References BR

Protein design: toward functional metalloenzymes.

How biology handles nitrite.

A copper-nitrosyl intermediate forms during the catalytic cycle of nitrite reductase, the enzyme that mediates the committed step in bacterial denitrification. The crystal structure of a type 2 copper-nitrosyl complex of nitrite reductase reveals an unprecedented side-on binding mode in which the nitrogen and oxygen atoms are nearly equidistant from the copper cofactor. Comparison of this structure with a refined nitrite-bound crystal structure explains how coordination can change between copper-oxygen and copper-nitrogen during catalysis. The side-on copper-nitrosyl in nitrite reductase expands the possibilities for nitric oxide interactions in copper proteins such as superoxide dismutase and prions.

https://www-ssrl.slac.stanford.edu/content/sites/default/files/documents/science-highlights/pdf/nir-200409.pdf

Side-on copper-nitrosyl coordination by nitrite reductase.

A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg The catalytic rate constants of all mutants were decreased to 04-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring

Kohichl Takagi

Papers

Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase.