Azurin aeruginosa

Antholine, W.E., Hanna, P.M., and McMillan, D.R. 1993. Low frequency EPR of Pseudomonas aeruginosa azurin analysis of ligand superhyperfine structure from a type 1 copper site. Biophysical Journal 64 267-272. 

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... 

Figure 39 X-Ray structure of the copper centre in azurin from Pseudomonas Aeruginosa...

Figure 40 Cyclic voltammograms recorded at a carbon paste electrode pretreated with 4,4-bipyridyl in an aqueous solution of azurin (from Pseudomonas Aeruginosa,) buffered at pH 7.0. Scans at different times (i) after 4 min (ii) after 13 min (iii) after 85 min...

For example, in Chapter 12, Section 4, we have examined the electrochemical response of azurin (from Pseudomonas aeruginosa), the only cupredoxin in which the copper(II) ion is pentacoordinate. Its reversible Cu(II)/Cu(I) reduction occurs at Eol= +0.31 V, vs. NHE, at 25° C. Measurements of the variation of the formal electrode potential with temperature in a non-iso thermic electrochemical cell gives the two diagrams illustrated in Figure ll.20... 

K. K. Turoverov, I. M. Kuznetsova, and V. N. Zaitsev, The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties, Biophys. Chem. 23, 79-89 (1985). 

Fig. 5. View of the blue copper and a5Ru(His83) centers in ruthenated Pseudomonas aeruginosa azurin [28]...

Intramolecular Ru(II) to Cu(II) ET rates have been measured in two other blue copper proteins, stellacyanin [42, 43] and azurin [9, 13, 28]. Pseudomonas aeruginosa azurin has been ruthenated at His83 [13] (Fig. 5). The intramolecular Ru(II) to Cu(II) ET rate of 1.9 s was found to be independent of temperature [28]. The Cu reorganization enthalpy was estimated to be < 7 kcal/mol [13, 28], a value confirming that blue copper is structured for efficient ET. Again, a blue copper ET rate is low in comparison with heme protein rates over similar distances (at similar driving forces) (Table 1). 

His35 to ligated His46 may be important in an electron transfer role or in His35 exercising some conformational control of the active site. The reduction potential of P. aeruginosa azurin increases from 300 mV (pH 8) to 360 mV (pH 5), which is believed to be related to His35 protonation. A pK of 6.6 is observed for this process, or alternatively pK s for azurin in the oxidized (6.1) and reduced (7.2) forms can be obtained [56]. 

It has long been known that, under some conditions at least, electron transfer between the c and d hemes of the P aeruginosa enzyme is slow and requires times of the order of seconds (22). What does this mean It is not necessarily related to the loss of the hydroxide ligand from the d heme iron, because under some experimental conditions used, azurin (a cupredoxin) was present and the enzyme was reduced at the outset,... 

It has long been assumed that azurin is an in vivo electron donor to cytochrome cdi of P. aeruginosa. The construction of mutants of P. aeruginosa in which one or both of the genes for azurin and cytochrome C551 have been deleted has led to the conclusion that in vivo cytochrome C551 is essential for the donation of electrons to the nitrite reductase and that azurin is ineffective (24). The discrepancy between in vivo and in vitro observations either could be reconciled if it is the failure of azurin to accept electrons from the cytochrome bc complex or another donor that is responsible for its ineffectiveness in vivo. 

The detailed mechanism of P aeruginosa CCP has been studied by a combination of stopped-flow spectroscopy (64, 65, 84, 85) and paramagnetic spectroscopies (51, 74). These data have been combined by Foote and colleagues (62) to yield a quantitative scheme that describes the activation process and reaction cycle. A version of this scheme, which involves four spectroscopically distinct intermediates, is shown in Fig. 10. In this scheme the resting oxidized enzyme (structure in Section III,B) reacts with 1 equiv of an electron donor (Cu(I) azurin) to yield the active mixed-valence (half-reduced) state. The active MV form reacts productively with substrate, hydrogen peroxide, to yield compound I. Compound I reacts sequentially with two further equivalents of Cu(I) azurin to complete the reduction of peroxide (compound II) before returning the enzyme to the MV state. A further state, compound 0, that has not been shown experimentally but would precede compound I formation is proposed in order to facilitate comparison with other peroxidases. 

The blue, or type 1, copper proteins, azurin from Pseudomonas aeruginosa (Adman et ai, 1978 Adman and Jensen, 1981) and from Al-caligenes denitrificans (Norris et al., 1983, 1986) and poplar plastocyanin (Guss and Freeman, 1983 Guss et al., 1986), have been studied by X-ray diffraction. These involve a Cu(I)/Cu(II) redox system. Cu(I) d ) is... 

Groeneveld etal. (1986) from an EXAFS study of Pseudomonas aeruginosa azurin, in which, on reduction, indications for a significantly shorter Cu-S bond were observed. 

A qualitative understanding of these features was provided in 1978 when the crystal structure of poplar leaf plastocyanin and Pseudomonas aeruginosa azurin appear-ed 2,73) proteins appears to be coordinated by two histidines with... 

Brunori, M., Parr, S. R., Greenwood, C., and Wilson, M. T. (1975). A temperature-jump study of the reaction between azurin and cytochrome c oxidase from Pseudomonas aeruginosa. Biochem. J. 151, 185-188. 

The azurins are electron-transfer proteins in the respiratory chains of certain bacteria. They have been particularly well studied from Pseudomonas aeruginosa and other pseudomonads, and contain one type 1 copper bound to a single polypeptide chain of molecular weight about 16 000. Amino acid sequence data for a number of azurins show that about one third of residues are conserved. All contain three cysteine residues. Three are also sequence homologies with the plastocyanins. 

Fig. 5.35. ID H NMR spectrum (200 MHz, 298 K) of Co(II)-azurin in H2O (adapted from [96]). A schematic drawing of the metal site in Pseudomonas aeruginosa native azurin is shown in the upper left comer.

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