Azurin characterization

The gene for this protein has also been isolated from A. faecalis and sequenced and expressed in Escherichia coli cells (Yamamoto et al., 1987). Like azurin, it contains an amino-terminal signal sequence, suggesting that it is secreted into the periplasm in A. faecalis, although not all of the blue protein is found in this fraction. Mutants of this protein are being made and characterized in T. Beppu s laboratory (Univ. of Tokyo) (personal communication, 1989). 

A recently characterized single-domain copper protein, auracyanin (Trost et al., 1988), is a dimeric protein which has a visible spectrum more like that of the A. faecalis cupredoxin (pseudoazurin, subgroup II see Table II) than that of either azurin or plastocyanin, but, because of its cysteine content and rhombic EPR, it has been put in the other class in Table II. 

The type-1 blue copper proteins act as electron carriers azurin, plastocyanin, stellacyanin, umecyanin e.g. They are characterized by a rather strong LMCT (ligand to metal charge transfer) band near 600 nm and by small hyperline coupling constants A in EPR. Copper is bound to two imidazole groups of histidine and to two... 

In this chapter, we overview first some recent examples of interfacial electrochemical ET of composite metalloproteins where molecular mechanistic detail has in some way been achieved. We discuss next some theoretical issues regarding in situ STM of large molecules, where resonance or environmentally activated tunnel channels are opened by the redox metal centre. This is followed by an overview of some recent achievements in the area of in situ STM/AFM of the single-metal proteins cytochrome c and azurin on polycrystalline and single-crystal platinum and gold surfaces. Such an integrated approach offers new perspectives for experimental and theoretical characterization of metalloproteins at solid surfaces in contact with the natural aqueous medium for metalloprotein function. 

The first class is cupredoxins—single-domain blue copper proteins composed of only one BCB domain. These proteins include plastocyanin, azurin, pseudoazurin, amicyanin, auracyanins, rusticyanin, halocyanin, and sulfocyanin (see Section IV). Plantacyanin of the phytocyanin family (Section V), subunit II of the cytochrome c oxidase, and the recently characterized nitrosocyanin also fall into this class. The last two are single BCB domain polypeptides closely related structurally to cupredoxins, but harboring, respectively, a binuclear copper site known as CuA and a novel type of copper-binding site called red (see Sections IX and X). 

Co(II) has been the most useful metal probe for the study of BCE The Co(II) derivatives of Ps. aeruginosa azurin (Moratal Mascarell et al., 1993a Piccioli et al., 1995 Salgado et al., 1995), Rhus vernicifera stellacyanin (Fernandez et al., 1997 Vila, 1994 Vila and Fernandez, 1996), Ac. cyclo-clastes pseudoazurin (Fernandez et al., 2001), Thi. ferrooxidans rusticyanin (Donaire et al., 2001), Thi. versutus amicyanin (Salgado et al., 1999), several mutants of azurin (Piccioli et al., 1995 Salgado et al., 1996, 1998a Vila et al., 1997), and the M99Q mutant of amicyanin (Diederix et al., 2000) have been prepared, and their H NMR spectra have been characterized. 

The type I copper sites function as electron transfer centers in the blue copper proteins and in multicopper enzymes, particularly oxidases (33). They are characterized by their intense blue color, their unusually small A values, and their very positive redox potentials (Table II). X-ray crystal structures of several blue copper proteins have been determined, notably plastocyanin (34), azurin (35), cucumber basic blue protein (36), and pseudoazurin (37). The active site structures show marked similarities but also distinct differences (Fig. 8). 

Some of the first protein systems where pulse radiolysis was used to help determine mechanism were those of blue copper proteins. These are proteins that are blue in solution and contain what are known as type (I) and type (2) copper centers. Two of the most well-known and well-characterized examples of these are azurin and cytochrome c. It was the studies of these systems that opened up the field of long-distance electron transfer in proteins and, by using the protein structure as a framework for electron transfer through space and through bonds, allowed for the development of a broad theoretical basis and many fascinating experiments on long-range electron transfer. Here, I will limit the discussion to electron transfer studies in azurin as illuminated by pulse radiolysis studies. ... 

Figure 2.12 Overview of three-dimensional structures and in situ STM images of metalloproteins representative of the three ET protein classes characterized by single-crystal PFV and in situ STM to single-molecule resolution, (a) Blue copper protein P. aeruginosa azurin (PDB 4AZU) [94] ...

Cyt c and azurin are structurally and in other respects very well characterized, and in-situ STM can be referred to many other structural, spectral, and kinetic data. No three-dimensional structure of laccase is available, but the structure of the closely related enzyme ascorbate oxidase is available with high resolution and srq ports a view of facile ET through die protein, involving all the copper atoms. 

Two features of the intramolecular LRET in azurins deserve attention. The first is that the copper site is characterized by an exceptionally low Frank-Condon barrier for electron transfer (14, 20). The second is that the Cys3-Cys26 disulfide bridge plays, most probably, only a structural role in this protein (20). Hence, the LRET induced from RSSR to the Cu(II) center is... 

Selectively modified and characterized ruthenated proteins have been prepared for cytochrome c (141-144), cytochrome c-551 (145), azurin (146, 147), plastocyanin (147), HiPlP (/4S), and myoglobin (77, 149-154). Intramolecular electron transfer rates, k, measured in these systems are listed in Table 111 along... 

Hasnain and coworkers have structurally and functionally characterized several mutants of NIR from Alcaligenes xylosoxidans. These smdies establish unequivocally that no direct ET occurs from the physiological electron donor (in this case azurin) to the catalytic type-2 copper center. The mutation of the axial type-1 copper ligand Metl44 to Leu increases both the redox potential and catalytic activity, establishing that the ratedetermining step of catalysis is the intermolecular ET from azurin to NIR. 

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