Redox active cofactors

A substantial fraction of the named enzymes are oxido-reductases, responsible for shuttling electrons along metabolic pathways that reduce carbon dioxide to sugar (in the case of plants), or reduce oxygen to water (in the case of mammals). The oxido-reductases that drive these processes involve a small set of redox active cofactors , that is, small chemical groups that gain or lose electrons. These cofactors include iron porjDhyrins, iron-sulfur clusters and copper complexes as well as organic species that are ET active. 

Figure 18.4 Structures of heme/Cu oxidases at different levels of detail, (a) Position of the redox-active cofactors relative to the membrane of CcO (left, only two obligatory subunits are shown) and quinol oxidase (right), (b) Electron transfer paths in mammalian CcO. Note that the imidazoles that ligate six-coordinate heme a and the five-coordinate heme are linked by a single amino acid, which can serve as a wire for electron transfer from ferroheme a to ferriheme as. (c) The O2 reduction site of mammalian CcO the numbering of the residues corresponds to that in the crystal structure of bovine heart CcO. The subscript 3 in heme as and heme 03 signifies the heme that binds O2. The structures were generated using coordinates deposited in the Protein Data Bank, lari [Ostermeier et al., 1997] Ifft [Abramson et al., 2000] (a) and locc [Tsukihara et al., 1996] (b, c).

Figure 13.9 (a) The structure of the four subunits of the CcO from R. sphaeroides (b) a more detailed view of the redox-active cofactors and amino acid residues in the proton transfer pathways (dotted arrows). (From Namslauer and Brzezinski, 2004. Copyright 2004, with permission from Elsevier.)... 

The enzymes of this type that have been characterized contain some type of redox-active cofactor, such as a flavin (3), or a metal ion (heme iron, non-heme iron, or copper), or both (4-6). Our understanding of the mechanism of these enzymes is most advanced in the case of the heme-containing enzyme cytochrome P450. But in spite of the availability of a crystal structure of an enzyme-substrate complex (7) and extensive information about related reactions of low molecular weight synthetic analogues of cytochrome P450 (8), a detailed picture of the molecular events that are referred to as "dioxygen activation" continues to elude us. 

Because metal ions bind to and modify the reactivity and structure of enzymes and substrates, a wide spectrum of techniques has been developed to examine the nature of metal ions which serve as templates, redox-active cofactors, Lewis acids/bases, ion-complexing agents, etc. 

Studies with the macrophage NOS were the first to show that NO synthesis was partially dependent on added Hbiopterin (Kwon et ai, 1989 Stuehr et al., 1990), which is a redox active cofactor utilized by the aromatic amino acid hydroxylases (Nichol et al., 1985). The requirement for H4biopterin has since been expanded to include all NOS isoforms studied to date. Mayer et al. (1990)... 

Figure 6 Galactose oxidase active site with its coordinated redox active cofactor.

IRadical cofactors in biological systems have become a subject of increasing interest in recent years (1-3). Tyrosine-based radicals, in particular, have now been identified in several enzymes (4). The tyrosine residue functions as a redox-active cofactor by interconverting between the oxidized phenoxyl radical and the normal phenol or phenolate states. More commonly known redox-active cofactors include transition metal ions, and a few enzymes use both tyrosine residues and metals as partners in effecting redox chemistry. 

Both amine and lysyl oxidases possess a single Type 2 copper center and a redox-active cofactor per subunit. As shown in Figure 18, amine oxidases contain 2,4,5-trihydroxyphenylalanine quinone (TPQ) and lysyl oxidase contains lysyl tyrosine quinone (LTQ), in which a lysine side chain has been cross-linked to TPQ. These cofactors are produced from the oxidation of a specific active-site tyrosine residue via novel self-processing events that require only copper and O2 see Metal-mediated Protein Modification). 

Covalent attachment has also been exploited for protein incorporation of non-native redox active cofactors. A photosensitive rhodium complex has been covalently attached to a cysteine near the heme of cytochrome c (67). The heme of these cytochrome c bioconjugates was photoreducible, which makes it possible for these artificial proteins to be potentially useful in electronic devices. The covalent anchoring, via a disulfide bond, of a redox active ferrocene cofactor has been demonstrated in the protein azurin (68). Not only did conjugation to the protein provide the cofactor with increased water stability and solubility, but it also provided, by means of mutagenesis, a means of tuning the reduction potential of the cofactor. The protein-aided transition of organometallic species into aqueous solution via increased solubility, stability and tuning are important benefits to the construction of artificial metalloproteins. 

Figure 3 The geometric arrangement of the redox active cofactors in Photosystem I and Photosystem II as given by the X-ray crystal structures PS I pdb entry IJBO (2) and PS II pdb entry 2AXT (3).

Semi-synthetic enzymes are produced by the reconstitution of apo-proteins with artificial active sites that yield novel catalytic functions [237]. For example, reconstitution of apo-myoglobin with Co(II)-protoporphyrin IX results in a novel biocatalyst that is capable of hydrogenating acetylene derivatives or evolving hydrogen [209, 238]. By the modification of the reconstitution of apo-proteins with artificial redox-active cofactors and the covalent attachment of photosensitizer units, photo-... 

Biological cell membranes are multi-component systems consisting of a fluid bilayer lipid membrane (BLM) and integrated membrane proteins. The main structural features of the BLMs are determined by a wide variety of amphiphilic lipids whose polar head groups are exposed to water while hydrocarbon tails form the nonpolar interior. The BLMs act as the medium for biochemical vectorial membrane processes such as photosynthesis, respiration and active ion transport. However, they do not participate in the corresponding chemical reactions which occur in membrane-dissolved proteins and often need redox-active cofactors. BLMs were therefore mostly investigated by physical chemists who studied their thermodynamics and kinetic behaviour . ... 

Electrons from cytochrome c are donated to the dinuclear copper centre Cua, and then transferred consecutively one at a time to haem a, and from there to the dinuclear haem-copper (haem u -Cua) catalytic centre. A tyrosine residue, Y(I-288), which is covalently cross-linked to one of the Cub ligands (His 240), is also part of the active site. The structure of the four-subunit CcO from R. spheroides is presented in Figure 13.9(a), while a more detailed view of the redox-active cofactors and amino acid residues involved in the proton transfer pathways is given in Figure 13.9(b) (Brzezinski Johansson, 2010). 

Redox-active cofactors are important species in biological systems, playing vital roles in redox and electron-transfer processes. Among the structurally and functionally diverse redox enzymes, flavoproteins containing the flavin cofactors flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) are involved in many different biochemical processes serving as a highly versatile redox... 

Figure 38 The redox-active cofactors of cytochrome c oxidase and the D and K proton transfer pathways (PDB-code 1M56), the red spheres are water moiecuies resoived in the X-ray crystai structures prepared with PyMOL (W. L. DeLano, Paio Alto, 2003).

Recent spectroscopic studies suggest, however, that this could happen only with the assistance of an active site lysyl residue [148]. Such a lysyl residue is found in the crystal structure of the active site of pea seedling amine oxidase [29], but is absent from E. coli amine oxidase [28]. The process of redox-active cofactor formation in phenethylamine oxidase and histamine oxidase of A. globiformis was recently analyzed by Raman spectroscopy using isotopic exchange. It was found that the oxygen on the... 

In conclusion, the experimental evidence currently available suggests that H4biopterin plays a dual role in NO synthesis. As an allosteric effector it appears to convert NOS in an active high-affinity conformation, and as a redox-active cofactor it may be involved in the protection of the enzyme... 

Figure 10.5 Putative pre-biotic chemistry routes Leading to the formation of anaerobic equivalents of the main purine and pyrimidine bases found in DNA and RNA. Intermediates formed also suggest pathways to anaerobic equivalents of redox active cofactors (see Chapter 4).

There are three very well studied DMSO reductase enzymes. The enzymes isolated from the purple photosynthetic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides (DorA) are periplasmic and share a high sequence identity. They are also the structurally simplest of all Mo enzymes ca. 85 kDa enzymes bearing a single redox active cofactor (the Mo active site). DMSO reductase from E. coli is a more complex membrane bound 140 kDa hetero-trimeric enzyme (DmsABC) bearing five Fe-S clusters in addition to the Mo active site. 

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