Azurin reduction potentials

It is an 0.2 2 tetramer of MW 115000. The larger of the two subunit types (MW 49000) each contain a covalently bound FAD that is the site of substrate oxygenation. The other subunits, c-type cytochromes of MW 8500, serve to relay electrons to an accepter molecule, which is probably azurin (reduction potential -I- 320 mV). In their electrochemical study. Hill and his group effectively provided an electrode surface as a substitute for this protein partner. 

A variety of physical methods has been used to ascertain whether or not surface ruthenation alters the structure of a protein. UV-vis, CD, EPR, and resonance Raman spectroscopies have demonstrated that myoglobin [14, 18], cytochrome c [5, 16, 19, 21], and azurin [13] are not perturbed structurally by the attachment of a ruthenium complex to a surface histidine. The reduction potential of the metal redox center of a protein and its temperature dependence are indicators of protein structure as well. Cyclic voltammetry [5, 13], differential pulse polarography [14,21], and spectroelectrochemistry [12,14,22] are commonly used for the determination of the ruthenium and protein redox center potentials in modified proteins. 

Studies on the bacterial type 1 protein azurin have been extensive. Ten different azurin amino-acid sequences have been determined with 47 out of 129 residues (Mf 14,(XX)) conserved. Reduction potentials are in the range 280 339 mV at... 

Earlier suggestions that the two uncoordinated and invariant residues His35 (inaccessible to solvent and covered by polypeptide) and His83 (remote and 13 A from Cu) are, from effects of [H ] on rate constants (and related pKg values), sites for electron transfer may require some re-examination. Thus, it has been demonstrated in plastocyanin studies [50] that a surface protonation can influence the reduction potential at the active site, in which case its effect is transmitted to all reaction sites. In other words, an effect of protonation on rate constants need not necessarily imply that the reaction occurs at the site of protonation. His35 is thought to be involved in pH-dependent transitions between active and inactive forms of reduced azurin [53]. The proximity of... 

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]. 

Mavicyanin (Mj = 18,000) is obtained from green squash (Cucurbito pepo medullosa), where it occurs alongside ascorbate oxidase [64]. It has a peak at 600 nm (e 5000 M cm and reduction potential of 285 mV. Further studies on this and the mung bean and rice bran proteins [65, 66] would be of interest. All the above type 1 Cu proteins have an intense blue color and characteristic narrow hyperfine EPR spectrum for the Cu(II) state. Table 3 summarizes the properties of those most studied. There is some variation in reduction potential and position of the main visible absorbance peak. In the case of azurin, for example, the latter is shifted from 597 to 625 nm. Stellacyanin has no methionine and the identity of the fourth ligand is therefore different [75]. The possibility that this is the 0(amide) of Gln97 has been suggested [63b]. It now seems unlikely that the disulfide is involved in coordination. Stellacyanin has 107 amino acids, with carbohydrate attached at three points giving a 40% contribution to the M, of 20,000 [75]. 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

Covalent attachment has also been exploited for protein incorporation of non-native redox active cofactors. A photosensitive rhodium complex has been covalently attached to a cysteine near the heme of cytochrome c (67). The heme of these cytochrome c bioconjugates was photoreducible, which makes it possible for these artificial proteins to be potentially useful in electronic devices. The covalent anchoring, via a disulfide bond, of a redox active ferrocene cofactor has been demonstrated in the protein azurin (68). Not only did conjugation to the protein provide the cofactor with increased water stability and solubility, but it also provided, by means of mutagenesis, a means of tuning the reduction potential of the cofactor. The protein-aided transition of organometallic species into aqueous solution via increased solubility, stability and tuning are important benefits to the construction of artificial metalloproteins. 

Garner DK, Vaughan MD, Hwang HI, Savelieff MG, Berry SM, Honek JE, Lu Y. Reduction potential tuning of the blue copper center in pseudomonas aeruginosa azurin by the axial methionine as probed by unnatural amino acids. J. Am. Chem. Soc. 2006 128 15608-15617. 

Moreover, other effects are as important as the ligands. The dielectric properties of the protein matrix are very different from those of water. It has often been argued that it behaves as a medium with a low dielectric constant (around 4 compared to 80 in water) [47,123,124]. Figure 11 shows that this gives rise to a very prominent change in the reduction potential of a blue-copper site [45]. It increases by 0.8-1 V as the site is moved from water solution to the centre of a protein with a radius of 1.5 nm (like plastocyanin) or 3.0 nm (like an azurin tet-ramer). It can also be seen that it is not necessary to move the site to the centre of the protein to get a full effect. Already at the surface of the protein, 80% of the maximum effect is seen, and when the site is 0.5 nm from the surface (as is typi-... 

Fig, 9. Variation of reduction potential with pH for the Pseudomonas aeruginosa azurin ACu(II)/ACu(I) couple from titrations with [FelCNlel ( ), and from rate constants for ACu(I) + [FelCNleP" and [FelCNle) " + ACu(II) ( ). 

Substitution of Se for S in Met 121 results in an increase in reduction potential of azurin fromP. aeruginosa by 30 mV (65). On changing Met 121 to Leu 121 by site-directed mutagenesis, the reduction potential is increased by 70 mV (66). Also by site-directed mutagenesis, replacement of the conserved Met 44 by Lys in the hydrophobic region results in a 40- to 60-mV increase in reduction potential (67). 

PascherT, Karlsson BG, Nordling M, Malmstrom BG, VanngardT (1993) Reduction potentials and their pH-dependence in site-directed-mutant forms of azurin from Pseudomonas-aeruginosa. Eur J Biochem 212(2) 289-296... 

Berry SM, Baker MH, Reardon NJ (2010) Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations. J Inorg Biochem 104 (10) 1071—1078... 

Lancaster KM, Sproules S, Palmer JH, Richards JH, Gray HB (2010) Outer-sphere effects on reduction potentials of copper sites in proteins the curious case of high potential type 2 C112D/M121E Pseudomonas aeruginosa azurin. J Am Chem Soc 132 14590-14595... 

The reduction potential of P. aeruginosa azurin increases by 60-70 mV upon changing pH from 10 to 5 (38 0). The NMR studies addressed the question of how much protonation of the two conserved titratable histidines in azurin, His35 and His83, may increase the Cu(ll)/Cu(l). It was found that the contributions from these two residues are 50 and 13 mV, respectively (40). There is a problem here, however, since the reduction potential of the H35K azurin mutant displays essentially the same pH dependence as WT (39), which seems to speak against this hypothesis, unless the protonated lysyl E-amine group has an unusually low pAT value. 

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