Rusticyanin

The amino acid sequences of both cytochromes c-552(s) (14 kDa) and c-552(m) (22.3 kDa) (Yano, 1992 Yamanaka and Fukumori, 1995) resemble those of cytochromes c2 and c4 (Meyer and Kamen, 1982). Cavazza et al. (1996) purified cytochrome c4 (21.2 kDa), which has two heme C molecules in the molecule. Cytochrome c-552(m) (22.3 kDa) seems to be identical with cytochrome c4 (21.2 kDa) on the basis of the amino acid sequence and molecular mass. The author and his colleagues were unable to find two molecules of heme C in the cytochrome c-552(m) (22.3 kDa) molecule. Giudici-Orticoni et al. (2000) reported that cytochrome c4 or two-heme cytochrome c-552(s) is intimately related to the oxidation of ferrous ion by rusti-cyanin. The cytochrome seems to be similar to the cytochrome c included in ferrous rusticyanin oxidoreductase (Blake and Shute, 1994). Furthermore, Appia-Ayme et al. (1998) claimed that as DNA that encodes cytochrome c-552(s) (14 kDa) was not found, this cytochrome could be a proteolytic product of cytochrome c4. However, they have not produced a distinct result showing that cytochrome c4 functions as the electron donor for cytochrome c oxidase like cytochrome c-552(s) (14 kDa). The reactivity with cytochrome c oxidase of cytochrome c-552(m) (22.3 kDa) or cytochrome c4 is very low in comparison to cytochrome c-552(s) (14 kDa) and its reaction with the oxidase is depressed by sulfate while that of cytochrome c-552(s) (14 kDa) is much accelerated by the salt, as already mentioned (Kai et al., 1992 Yamanaka and Fukumori, 1995). These results seem to argue against cytochrome c-552(s) (14 kDa) being a proteolytic product of cytochrome c-552(m) (22.3 kDa)or cytochrome c4. 

Rusticyanin is a copper-containing blue protein. It was first purified by Cox and Boxer (1978). The author and his colleagues purified it to a homogeneous state and 

Cytochrome c oxidase of Acidithiobacillus ferrooxidans was previously called cytochrome a1( as it shows the a peak at 595 nm (Ingledew, 1982). However, as the oxidase purified from the bacterium has two heme A molecules and two copper atoms in the minimal functional unit and one of the two molecules of heme A combines with carbon monoxide, it is a cytochrome aa3-type cytochrome c oxidase although it has the a peak at 595 nm (Kai et al., 1992). It differs from the usual cytochrome aa3 in having only one molecule of heme A and one atom of copper in the minimal structural unit, which comprises one molecule each of three kinds of subunits (54 kDa, 21 kDa, 15 kDa) like Starkeya novella cytochrome c oxidase (Shoji et al., 1992). [The DNA study suggests the presence of four subunits with the molecular masses of 69, 28, 18 and 6.4 kDa (Appia-Ayme et al., 1999)]. The minimal functional unit of the A. ferrooxidans oxidase is a dimer of the minimal structural unit, and the dimer shows general properties of cytochrome ach except that the a peak is present at a wavelength shorter than 600 nm of the absorption spectrum. The oxidase resembles Nitrosomonas europaea cytochrome c oxidase (Yamazaki et al., 1985) (see pp. 25-26) in the position of the a peak of the absorption spectrum. 

ferrooxidans oxidase catalyzes the oxidation of ferrocytochrome c-552(s) (14 kDa), ferrocytochrome c-552(m) (22.3 kDa) or ferrocytochrome c4, and ferrocytochrome c-550(m) (51 kDa). Although the oxidase also catalyzes the oxidation of the reduced form of rusticyanin, its Km for rusticyanin is fairly large (600 pM) (Kai et al., 1992 Yamanaka and Fukumori, 1995) while Kms of the oxidase for cytochromes c-552(s), c-552(m) and c-550(m) are 17, 2.2, and 4.2 jM, respectively. However, as the concentration of rusticyanin in the bacterial cells is considerably high, the copper protein can also be the electron donor for the oxidase in vivo. Although the oxidase shows a catalytic capability to oxidize molybdenum blue (Sugio et al 1992a), the catalysis is not limited to the A. ferrooxidans oxidase because some oxidases belonging to cytochrome aa3 also show such activity (Kai et al., 1992). 

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Several copper enzymes will be discussed in detail in subsequent sections of this chapter. Information about major classes of copper enzymes, most of which will not be discussed, is collected in Table 5.1 as adapted from Chapter 14 of reference 49. Table 1 of reference 4 describes additional copper proteins such as the blue copper electron transfer proteins stellacyanin, amicyanin, auracyanin, rusticyanin, and so on. Nitrite reductase contains both normal and blue copper enzymes and facilitates the important biological reaction NO) — NO. Solomon s Chemical Reviews article4 contains extensive information on ligand field theory in relation to ground-state electronic properties of copper complexes and the application of... 

Since rusticyanin is positively charged (p/=9.1), its electrochemical response should be tested at negatively charged electrodes (namely, edge-plane pyrolitic graphite or glassy carbon electrodes). As a matter of fact, no positive response is obtained at a carbon paste electrode, but in the presence of a promoter such as 4,4/-bipyridyl a sufficiently resolved cyclic voltammetric response is obtained, Figure 37.67... 

In agreement with the chemical reversibility of the Cu(II)/Cu(I) reduction, the reduced form of rusticyanin possesses a copper coordination quite similar to that of the oxidized form.69... 

Figure 37 Cyclic voltammogram recorded at a carbon paste electrode pretreated with 4,4 -bipyridyl in an aqueous solution (pH 2.2) of rusticyanin...

There are four other proteins - stellacyanin, rusticyanin, umecyanin and ami-cyanin (Table 3) which have been fairly extensively studied. A crystal structure determination for amicyanin from Thiobacillus versutus is now under way [61]. A number of other type 1 proteins have been identified. These include pseudo-... 

Rubredoxin(s) 858[s ], 859 Fe-S cluster 857s Ruhemann s purple 121s Rusticyanin 883 Rusts 20... 

The other small blue proteins are only poorly characterized at present It is assumed their function is that of electron transfer. Rusticyanin from Thiobacillus ferrooxidans is thought to be the initial electron acceptor from iron(II) in the respiratory chain at pH 2. Rusticyanin contains 159 residues, with one cysteine, three methionine and five histidine residues. The protein is unusually stable at low pH, in accord with its presence in an acidophilic organism. The midpoint potential of rusticyanin is high (+680 mV), and is second in magnitude only to that of Polyporus laccase. 

Reduction of rusticyanin by iron(II) and by chromium(II) has been reported.94Sa The former reaction shows effects due to anions, with limiting zero order kinetics observed in the presence of sulfate. 

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). 

Little is known about the redox partners of rusticyanin, although a diheme cytochrome, cyt c4, has been imphcated because of its abihty to form a complex with rusticyanin. The complex formation between these two proteins at pH 4.8 induces a dramatic decrease, by almost 100 mV, in the redox potential of rusticyanin, while the potentials of both hemes in the cytochrome remain unchanged. Interestingly, complex formation is also accompanied by changes in the electronic absorption spectrum of rusticyanin that are reminiscent of those observed for the uncomplexed protein at high pH (above pH 7.0) (Giudici-Orticoni et al., 1999). 

Fig.2. Schematic drawing of the metal coordination sites in typical Type I copper proteins (A) plastocyanin (B) rusticyanin (C) stellacyanin and (D) azurin.

Rusticyanin is stable down to pH 2, and no change in the ESE rate is observed over a broad pH range (Kyritsis et al., 1995). This is consistent with the hydrophobic nature of the residues that surround the active site. The lower ESE rate compared to that of azurin (cf. Table III) is suggested to be related to the longer copper-copper distance due to a more deeply buried metal site (Kyritsis et al., 1995). 

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. 

In rusticyanin and pseudoazurin, the axial Met ligand adopts a different orientation than in azurin, resulting in a stronger Cu(II)-S8 Met bond and a tetragonal distortion. The magnetic anisotropy tensor is rhombic in the Co(II)-substituted proteins, and the pseudocontact contribution to... 

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