Azurin source

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

Intriguingly, the blue copper sites, especiaUy those with a carbonyl oxygen at the axial coordination position, display high affinity for Zn + ions. Mutants in which the Met is replaced by Gin or Glu preferentiaUy bind Zn + when expressed in heterologous systems, e.g., Escherichia coli. Examples include azurin, amicyanin, nitrite reductase, and possibly also plastocyanin (Diederix et al., 2000 Hibino et al., 1995 Murphy et al., 1995 Nar et al., 1992a Romero et al., 1993). In the case of azurin it has been shown that both wild-type and the Met—Gin mutant have the same affinity for both Zn +and Cu + (Romero ci a/., 1993). In addition, EXAFS studies showed that some preparations of blue copper proteins purihed from their natural sources also contain small fractions of Zn derivatives (DeBeer George, personal communication). 

There is a third class of peroxidases isolable from the bacterial sources such as P. aeruginosa and Pseudomonas stutzerii, which oxidize cytochrome C551, or azurin. This protein contains two heme protoporphyrin IX groups covalently bound to a single polypeptide side chain. In this enzyme one heme group is oxidized from Fe(III) to Fe(IV)=0 and the second heme, from Fe(II) to Fe(III). The oxidizing equivalents are directed to two centers with very different redox potentials (75). 

Direct electrochemistry has also been used (72-78) to couple the electrode reactions to enzymes for which the redox proteins act as cofactors. In the studies, the chemically reduced or oxidized enzyme was turned over through the use of a protein and its electrode reaction as the source or sink of electrons. In the first report (72, 73) of such application, the electrochemical reduction of horse heart cjd,ochrome c was coupled to the reduction of dioxygen in the presence of Pseudomonas aeruginosa nitrite reductase/cytochrome oxidase via the redox proteins, azurin and cytochrome C551. The system corresponded to an oxygen electrode in which the four-electron reduction of dioxygen was achieved relatively fast at pH 7. 

Figure 10.1 Cone-axis oscillation photographs of an azurin crystal recorded (a) at Stanford on SSRL (A=l.740 A), and (b) with a Cu Ka sealed tube X-ray source. The SR-based data extended to higher resolution. Extracted from Phillips et al (1976) with permission.

Proteins of this class which have received the most attention were isolated from four bacterial species Pseudomonas aeruginosa, Ps. fluorescens, Ps. denitri-ficans, and Bordetella pertussis, although Sutherland (5) has isolated azurin from several other strains of Pseudomonas, Bordetella, and Alcaligenes. The near identity of azurins isolated from these different sources, has been generally assumed, and with one possible exception, that from Ps. denitrificans which does not bind to carboxy-methyl cellulose resin at the same pH as the other proteins (5), this seems to be the case. Ambler and Brown [6), who have elucidated the amino acid sequence of Ps. fluorescens azurin state that B. bronchiseptica, A. denitrificans and faecalis, Ps. denitrificans and fluorescens yield azurins having homologous amino acid sequences. 

General Chemical Properties. The information assembled in Table 1 is consistent with azurins from the several sources indicated containing a single Cu atom in a protein of molecular weight approximately 16,000 and very little if any associated carbohydrate. 

Cross reactions of plastocyanins and azurins from various sources with cytochromes can be fitted to the Marcus equation using constant self-exchange rate constants of 6.6 x 10 s for the plastocyanins, 9.9 x 10 s for Ps. 

Azurin a family of blue, copper-containing proteins from Pseudcmumas, Alcattgenes and Bordetelia species. A. from Pseudorrumas fluorescens contains 128 amino add residues of known sequence, and one intrachain disulfide bond. A. from all sources has M, 14,000-16,000 and homologous primary and tertiary structure, but different spedes of A. have different redox potentials. 

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