Metalloprotein classes

Lactamases (EC 3.5.2.6) inactivate /3-lactam antibiotics by hydrolyzing the amide bond (Fig. 5.1, Pathway b). These enzymes are the most important ones in the bacterial defense against /3-lactam antibiotics [15]. On the basis of catalytic mechanism, /3-lactamases can be subdivided into two major groups, namely Zn2+-containing metalloproteins (class B), and active-serine enzymes, which are subdivided into classes A, C, and D based on their amino acid sequences (see Chapt. 2). The metallo-enzymes are produced by only a relatively small number of pathogenic strains, but represent a potential threat for the future. Indeed, they are able to hydrolyze efficiently carbape-nems, which generally escape the activity of the more common serine-/3-lac-tamases [16] [17]. At present, however, most of the resistance of bacteria to /3-lactam antibiotics is due to the activity of serine-/3-lactamases. These enzymes hydrolyze the /3-lactam moiety via an acyl-enzyme intermediate similar to that formed by transpeptidases. The difference in the catalytic pathways of the two enzymes is merely quantitative (Fig. 5.1, Pathways a and b). 

HEMOPROTEINS. These proteins are actually a subclass of metalloproteins because their prosthetic group is heme, the name given to iron protoporphyrin IX (Figure 5.15). Because heme-containing proteins enjoy so many prominent biological functions, they are considered a class by themselves. 

Classes of metalloproteins. Transition ion prosthetic groups in proteins are... 

Zinc is the active metal in the largest group of metalloproteins found in the nature. Recently a new class of zinc enzymes with a sulfur-rich environment has emerged the thiolate-alkylating enzimes, the most prominent of which is the cobalamine-independent methionine synthase.126 For these reasons several monothiolate zinc complexes have been prepared for the modelling of these enzymes with different N2S as (13),127 130 N20,13° 132 N3,132,133 S3,134 tripod ligands, or with Cd because of the favourable spectroscopic properties with an S3 tripod ligand.135... 

Chapter 6). Other iron-sulfur proteins, so named because they contain iron sulfur clusters of various sizes, include the rubredoxins and ferredoxins. Rubredoxins are found in anaerobic bacteria and contain iron ligated to four cysteine sulfurs. Ferredoxins are found in plant chloroplasts and mammalian tissue and contain spin-coupled [2Fe-2S] clusters. Cytochromes comprise several large classes of electron transfer metalloproteins widespread in nature. At least four cytochromes are involved in the mitrochondrial electron transfer chain, which reduces oxygen to water according to equation 1.29. Further discussion of these proteins can be found in Chapters 6 and 7 of reference 13. 

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

Three main classes of metalloproteins able to coordinate reversibly dioxygen are known, namely ... 

The use of XAS for structural characterization of the metal sites in metalloproteins has increased dramatically in the last ten years. Throughout most of this period, advances in biological XAS have bren driven largely by the increasing availability of synchrotron ra ation and by the increasing quality of synchrotron sources. With each new increase in the available synchrotron intensity, new classes of experiments have become feasible. Some of the inherent limitations of the XAS method have already been discussed. It is appropriate now to consider more practical concerns regarding the future of biological XAS, and the way in which this will be affected by the development of new synchrotron sources. 

Before plunging into a discussion of how such complexes are prepared, it is perhaps worthwhile to consider explicitly the rationale for such activity. The synthesis and characterization of accurate model complexes for a given metal site in a protein or other macromolecule allows one to (l) determine the intrinsic properties of the metal site in the absence of perturbations provided by the protein environment or (il) in favorable cases, deduce the structure of the metal site by comparison of corresponding physical and spectroscopic properties of the model and metalloprotein (3). The first class of model complexes has been termed "corroborative models" by Hill (4), while the second are termed "speculative models" (4). To date, virtually all the major achievements of the synthetic model approach have been in development of corroborative models. 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

In the preceding sections, consideration has been given to important classes of metalloproteins which are involved in electron transfer, namely cytochromes, iron-sulfur proteins and the blue... 

Our discussion of scanning probe approaches to metalloproteins at surfaces indicates, first that in situ STM and AFM mapping of metalloproteins with molecular (rather than atomic) resolution is definitely feasible. In addition, submolecular detail is possibly within reach. This is interesting as proteins constitute a class of soft materials. It is also comforting as aqueous solution is otherwise the natural functional medium for proteins. 

Stellacyanin, the plastocyanins, and the azurins are the most widely studied copper-containing metalloproteins of the next active-site class, the Blue Copper sites. These proteins, which generally appear to be involved in redox chemistry, have quite unique spectral features32,33). The potential for complementary interaction between inorganic spectroscopy and protein crystallography is well demonstrated by the roles that they have played in generating fairly detailed geometric and electronic structural pictures of the Blue Copper metal centers. 

Heme proteins are one of the largest classes of metalloproteins studied to date see Iron Heme Proteins Dioxygen Transport Storage Iron Heme Proteins, Peroxidases, Catalases <6 Catalase-peroxidases). More than 5% of protein structures in the Protein Data Bank contain at least one heme moiety. It is no wonder that designing heme proteins has been one of the most active areas of research. Since the dominant secondary structure in heme proteins is a-hehces (accounting for 77% of all secondary structures in known heme proteins),a number of Q -helix-containing metalloporphyrin-peptides have been synthesized using either covalent or noncovalent approaches. 

Some metal ions in metalloproteins retain a normal metal environment, behave as classical coordination complexes, and are less studied because they appear to be understood (although perhaps this is a smaller class than it once appeared). The particular properties of other sites can often be traced back to unusual geometry or ligation at the active site these systems have generally been more studied because they are more intriguing. 

As stated above, the electronic spectra of the Class III mixed-valence biological sites have been interpreted in terms of sulfur ligation. However, similar intense features are observed in the visible and MCD spectra of mixed-valence complexes of L44 and L45, which contain no sulfur donors. Further, the presence of essentially identical spectra for model complexes with imine donors and with amine donors suggests that the absorption is not due to charge transfer involving the donor atoms. These bands are therefore ascribed to electronic transitions within the [Cu(1.5)—Cu(1.5)] unit and this may suggest an alternative explanation for the metalloprotein spectra. 

Metalloproteins, where the active site includes one or more metals, represent a very different class of proteins than those discussed above. The particular kinds of metalloproteins discussed here are those where the metal is redox active and represents a functional and not structural component of the system. Many mechanistic studies of metalloproteins have been carried out using radiation chemistry in the past 50 years. Two different ways of using radiation chemistry to query mechanisms will be illustrated here. The first, as described in the earliest of these studies using blue copper proteins such as azurin, involves using pulse radiolysis to change an oxidation state and thus... 

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