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The cytochrome P450 superfamily of heme monooxygenases catalyse many reactions involved in the biosynthesis and degradation of endogenous compounds and in the oxidative metabolism of xenobiotics. These enzymes all share the same overall catalytic mechanism, and their varied substrate specificities arise from differences in their active site topologies, suggesting that it should be possible to engineer P450 enzymes for the oxidation of unnatural substrates. A review of the active site protein engineering experiments on cytochrome P450cam reported to date is presented here. Cytochrome P450cam is a stable, soluble enzyme isolated from Pseudomonas Putida, and is an excellent system for rational protein engineering. The current understanding of the details of the P450cam catalytic mechanism, including the uncoupling pathways, is first discussed. The factors controlling substrate access, binding and turnover activity, and the coupling efficiency have been investigated by site-directed mutagenesis. The results of this work have enabled the rational redesign of P450cam to broaden the substrate range, to improve the activity and coupling efficiency towards unnatural substrates, and to engineer the regioselectivity of substrate oxidation. The construction of a "self-sufficient" fusion protein containing all three proteins of the P450cam system brings one step nearer the in vitro and in vivo biotechnological applications of P450cam. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases catalyze an essential reaction in all living cells — the production of deoxyribonucleotide precursors for DNA synthesis. An intricate allosteric regulation enables one and the same enzymic component to accept both purine and pyrimidine ribonucleotide substrates and to furnish growing cells with balanced deoxyribonucleotide pools. The reaction, which is based on radical chemistry, is common to all ribonucleotide reductases, whereas the mode of furnishing the radical differs and is currently a basis for distinguishing at least three different classes of ribonucleotide reductases. The radical generating components, although of seemingly different evolutionary origin, all include transition metal binding sites. Studies on the ribonucleotide reductase family highlight exciting mechanistic and evolutionary implications of far-reaching significance. [ABSTRACT FROM AUTHOR]

Structural, spectroscopic and chemical features of the metal centres of non-haem-iron-containing cytochrome b557 are described. This haemoprotein is a member of the ferritin family of proteins and thus is also called bacterioferritin. It consists of 24 polypeptide subunits and, in addition to its inter-subunit bis-methionine coordinated haem b groups, it also contains intra-subunit dinuclear metal centres and has the capacity for a central non-haem-iron deposit of 4,500 Fe(III) ions per molecule. The dinuclear site can be occupied by Co(II) or Mn(II), and probably by Zn(II) and Fe(II) as well. Mixed metal centres also appear to be formed. The non-haem-iron core can be either amorphous, as customarily found with native proteins, or crystalline. Crystalline cores can be laid down in vitro. This latter type of core is often superparamagnetic and this phenomenon is decribed, particularly with regards to its characterisation by 57Fe Mössbauer spectroscopy, EPR spectroscopy and Magnetic circular dichroism spectroscopy. Finally, possible functional roles for the metal centres are described. [ABSTRACT FROM AUTHOR]

Serum transferrin is an 80 kDa glycoprotein which transports Fe3+ and a variety of metal ions of diagnostic, therapeutic and toxic importance. Methods for the study of the differences between the two metal binding sites (the N- and C-lobe sites), and mechanisms for the uptake and release of both metal ions and synergistic anions are discussed. The strength of metal binding to transferrin can be rationalised on the basis of metal ion acidity (strength of hydroxide binding). Similarly the strength of metal binding to the enzymes carbonic anhydrase and carboxypeptidase can be correlated with the binding of the same metal ions to imidazole. Such correlations of metal binding to proteins and low molecular mass ligands may provide insight into the preorganisation of metal binding sites in proteins. [ABSTRACT FROM AUTHOR]

Iron-porphyrin complexes, known as hemes, form the prosthetic groups of a number of proteins. These hemoproteins exhibit an impressive range of biological functions. These include: Simple electron transfer reactions, oxygen transport and storage, oxygen reduction to the level of hydrogen peroxide or water, oxygenations of organic substrates, and the reduction of peroxides. This diversity of function is often extended further by combining heme groups with other cofactors, e. g. flavins and/or metal ions. Such combinations frequently allow heme cofactors to couple electron transfers with other processes, such as the translocation of protons or the reduction/oxidation of other molecules. This versatility in function is made possible by a combination of differences in both the polypeptide and heme constituents of the various hemoproteins. The aim of this article is to illustrate how nature has used different protein frameworks to exploit the redox properties of heme. This is done by focusing on a carefully chosen selection of hemoproteins which exemplify the numerous redox functions performed by heme in biology. [ABSTRACT FROM AUTHOR]

Iron biominerals are ubiquitous and fulfil many functions. These are often iron oxide, oxyhydroxide and hydroxide phases, many of which are common in the lithosphere, but some of which, such as the mineral found within the iron storage protein ferritin, are metastable. This article takes the specific example of loaded ferritin as a biomineral and explores the use of model compounds to elucidate the formation, structure and properties of the iron oxyhydroxide mineral contained within the protein. The utility of various compounds is evaluated by considering how closely they match a number of essential criteria defining the composition and properties of loaded ferritin. Those compounds with well-defined structures which can be regarded as "scale models" of the loaded ferritin protein are found to be the most realistic mimics. [ABSTRACT FROM AUTHOR]

Biological chemistry is a major frontier of inorganic chemistry. The three special volumes of Structure and Bonding devoted to Metal Sites in Proteins and Models address the questions: how unusual (“entatic”) are metal sites in metalloproteins and metalloenzymes compared to those in small coordination complexes? And if they are special, how do polypeptide chains and co-factors control this? The chapters deal with iron, with metal centres acting as Lewis acids, metals in phosphate enzymes, with vanadium, and with the wide variety of transition metal ions which act as redox centres. They illustrate in particular how the combined armoury of genetics and structure determination at the molecular level are providing unprecedented new tools for molecular engineering. A steady flow of reports over the last seven years on the molecular characterization of tungsten-containing proteins from a wide range of microorganisms has drastically changed our appreciation of tungsten in redox enzymes. Biological tungsten is not an odd remnant of evolution but a widespread, versatile catalytic entity for the activation of the carbonyl group both in carbon dioxide and in a broad spectrum of aldehydes and carboxylic acids. This review starts off with an outline of the boundary conditions for the biological use of tungsten dictated by inorganic chemistry. Subsequently, the possibilities for spectroscopy on tungsten proteins are reviewed. The molecular properties of tungsten enzymes are described and are related to their physiology and their mechanism of action. Gaps in our current knowledge of the bio-inorganic chemistry of tungsten are identified, and possible directions of future research are indicated. [ABSTRACT FROM AUTHOR]

Biological chemistry is a major frontier of inorganic chemistry. The three special volumes of Structure and Bonding devoted to Metal Sites in Proteins and Models address the questions: how unusual (“entatic”) are metal sites in metalloproteins and metalloenzymes compared to those in small coordination complexes? And if they are special, how do polypeptide chains and co-factors control this? The chapters deal with iron, with metal centres acting as Lewis acids, metals in phosphate enzymes, with vanadium, and with the wide variety of transition metal ions which act as redox centres. They illustrate in particular how the combined armoury of genetics and structure determination at the molecular level are providing unprecedented new tools for molecular engineering. The aim of this article is to critically discuss the information available on the influence of the protein environment on the electron transfer properties of Fe-S proteins. First, the intrinsic redox and structural features of synthetic Fe-S clusters are illustrated, in order to provide a background on which the effects of the protein matrix can be subsequently highlighted. The parameters found to influence the reduction potential in metalloproteins are then discussed, and a detailed analysis of structure-function relationships among the different classes of Fe-S proteins is carried out. This analysis reveals that, within each class, one of the parameters is often more important than the others in determining the reduction potential of the Fe-S center; the parameter changes upon passing from one class to the other. Within the class of high potential Fe-S proteins the major determinant for the variation of reduction potential is the difference in charged residues, whereas in rubredoxins and [2Fe-2S], [3Fe-4S], and [4Fe-4S] ferredoxins, this effect is attenuated and overwhelmed by solvent accessibility to the cluster core and/or inner-fractional charges in the protein itself. A deeper understanding of the influence of the structural properties of the protein is gained by analysing the microscopic reduction potentials of the individual Fe ions in Fe-S centers. The latter are obtained from the electronic distribution within each type of cluster core, which, in turn, is determined from a theoretical analysis of the electron-nucleus coupling derived from NMR and other spectroscopic analysis. [ABSTRACT FROM AUTHOR]

Biological chemistry is a major frontier of inorganic chemistry. The three special volumes of Structure and Bonding devoted to Metal Sites in Proteins and Models address the questions: how unusual (“entatic”) are metal sites in metalloproteins and metalloenzymes compared to those in small coordination complexes? And if they are special, how do polypeptide chains and co-factors control this? The chapters deal with iron, with metal centres acting as Lewis acids, metals in phosphate enzymes, with vanadium, and with the wide variety of transition metal ions which act as redox centres. They illustrate in particular how the combined armoury of genetics and structure determination at the molecular level are providing unprecedented new tools for molecular engineering. Hydrogenases are proteins which catalyze the reversible two-electron oxidation of the most simple of chemical compounds, molecular hydrogen, following the reaction: H2 ⇔ 2H++2e−. These enzymes are the central feature of hydrogen metabolism which is essential to many microorganisms of great biotechnological interest, such as methanogenic, acetogenic, nitrogen-fixing, photosynthetic and sulfate-reducing bacteria. For the last twenty years hydrogenases have enjoyed renewed interest, mostly in view of their capacity to produce molecular hydrogen, a clean source of energy. The determination of the first three-dimensional atomic structure of a hydrogenase by single crystal X-ray diffraction, together with infrared (IR) and electron paramagnetic resonance (EPR) spectroscopy have revealed that the active site consists of a bimetallic center containing for [NiFe] hydrogenases one nickel and one iron atom and three non-protein diatomic ligands (one CO and two CN−) to the iron. This evidence with stoichiometric redox-titrations has led to new insights into the catalytic mechanism. Moreover, genetic studies have cast light on the biosynthesis of the protein, including the incorporation of metal atoms found at the active site. A brief overall view of the structural and functional properties of hydrogenase is presented. The crystal structure is described with emphasis on the determination of the atomic content and arrangement of the active site from combined X-ray and spectroscopic data. Some aspects of the catalytic mechanism are discussed in the light of these new results. [ABSTRACT FROM AUTHOR]

Biological chemistry is a major frontier of inorganic chemistry. The three special volumes of Structure and Bonding devoted to Metal Sites in Proteins and Models address the questions: how unusual (“entatic”) are metal sites in metalloproteins and metalloenzymes compared to those in small coordination complexes? And if they are special, how do polypeptide chains and co-factors control this? The chapters deal with iron, with metal centres acting as Lewis acids, metals in phosphate enzymes, with vanadium, and with the wide variety of transition metal ions which act as redox centres. They illustrate in particular how the combined armoury of genetics and structure determination at the molecular level are providing unprecedented new tools for molecular engineering. The coordination geometries of metal sites in cupredoxins, mutants and metal derivatives of cupredoxins, multi-copper oxidases and a vanadium-containing chloroperoxidase as derived from X-ray crystallography are described. Correlations with their spectroscopic, electrochemical, electron transfer and catalytic properties are discussed. X-ray crystallography, EPR and Resonance Raman spectroscopy of copper sites in cupredoxins and mutants have led to a classification ranging from type 1 trigonal, type 1 distorted tetrahedral, type 1.5 to type 2. The mutation of copper ligands in azurin or amino acids close to the copper site changes the redox potential in a range of ± 140 mV, only. The high redox potential of rusticyanin of 680 mV (azurin, 380 mV) should be mainly due to the special protein environment of the copper site (high proportion of hydrophobic residues). The type 1 and trinuclear copper centres of the multi-copper oxidases ascorbate oxidase, laccase and ceruloplasmin are presented. The metal sites of type 2 depleted, fully-reduced, peroxide and azide forms of ascorbate oxidase, as determined by X-ray crystallography, are discussed in terms of the mechanistic properties of these enzymes. The first X-ray structure of a vanadium-containing protein, namely of a chloroperoxidase from the fungus Curvularia inaequalis, is briefly discussed. The protein fold is mainly α-helical with two four-helix bundles. In the X-ray structure, which is an azide:enzyme complex, the vanadium exhibits a simple unexpected coordination geometry, namely, a trigonal bipyramidal coordination with three non-protein oxygen ligands (VO3 group), one nitrogen ligand from a histidine and one nitrogen from the exogenous azide ligand. [ABSTRACT FROM AUTHOR]

Biological chemistry is a major frontier of inorganic chemistry. The three special volumes of Structure and Bonding devoted to Metal Sites in Proteins and Models address the questions: how unusual (“entatic”) are metal sites in metalloproteins and metalloenzymes compared to those in small coordination complexes? And if they are special, how do polypeptide chains and co-factors control this? The chapters deal with iron, with metal centres acting as Lewis acids, metals in phosphate enzymes, with vanadium, and with the wide variety of transition metal ions which act as redox centres. They illustrate in particular how the combined armoury of genetics and structure determination at the molecular level are providing unprecedented new tools for molecular engineering. The photosynthetic conversion of solar to chemical energy is based on light-driven charge separation in a chlorophyll-based pigment. In higher-plants, the electrons required for this process are extracted from H2O, ultimately producing O2 as a waste by-product of photosynthesis. The photosynthetic oxidation of water takes place at the oxygen evolving complex (OEC) on the donor (lumenal) side of Photosystem II. The OEC contains four Mn ions, together with calcium and chloride as essential inorganic cofactors. The techniques which have proven most useful in characterizing the nature of the OEC are X-ray absorption spectroscopy and EPR. Recent results from both techniques are reviewed. [ABSTRACT FROM AUTHOR]