Chapter 7: The role of copper ligands on electron transfer in azurins

The long-range intramolecular electron transfer (ET) rates between the disulfide radical anion and the copper site in azurin from Alcaligenes denitrificans and three mutants of azurin from A. denitrificans and Pseudomonas aeruginosa have been measured. In the three mutants a copper ligand of azurin was substituted by a glycine (His46Gly and His117Gly azurin) or a histidine (Met121His azurin). Met121His azurin exhibits an ET rate at 298 K which is half that of wt azurin from A. denitrificans (21±4 vs. 42±4 s). His46Gly and His117Gly azurin both exhibit slower ET rates (15±2 and 7±3 s, resp.) compared to wt azurin from P. aeruginasa (44±7 s). When the oxidised copper site of His117Gly azurin is reconstituted with imidazole the ET rate increases to 149±17 s. From the temperature dependence the activation enthalpy and entropy have been determined. The ET rates are discussed in terms of the Marcus equation. The similar ET rates of His46Gly and His117Gly suggests that ET does not proceed via a pathway that includes the imidazole ring of His46. The importance of a pathway via the internally positioned tryptophan is discussed taking into account the observed enthalpy-entropy compensation for a range of azurins (mutants). Electron tranfer in azurin mutants 116 Introduction Electron transfer (ET) plays an important role in many biological systems and a central question is whether specific structural features of proteins promote ET [Beratan et al., 1992; Broo et al., 1992; Farid et al., 1993; Gray et al., 1996; Moser et al., 1992; Siddarth et al., 1993]. The blue copper protein azurin has become an effective model for examination of intramolecular long range ET (LRET) in proteins [Farver et al., 1989; Farver et al., 1992a; Farver et al., 1992b; Farver et al., 1993; Farver et al., 1996a; Farver et al., 1996b; Farver et al., 1997b]. It consists of a rigid β-sheeted polypeptide, and threedimensional structures have been determined for a large number of wild type (WT) and single site mutated azurins [Baker, 1988; Hammann et al., 1996; Nar et al., 1991b; Nar et al., 1991a; Romero et al., 1993]. Furthermore, in order to study internal ET, no modification by an external redox group is needed, since azurin contains two potential redox centres, (1) the copper ion coordinated directly to amino acid residues and (2) a disulfide bridge (RSSR) at the opposite end of the molecule. It has been previously demonstrated that LRET between these two centres can be induced by pulse radiolytic reduction of RSSR [Farver et al., 1989; Farver et al., 1992a; Farver et al., 1992b; Farver et al., 1993; Farver et al., 1996a; Farver et al., 1996b; Farver et al., 1997b]. Using both wild type and single-site mutated azurins, the effect of specific structural changes on the rate of intramolecular ET has been studied. Blue copper proteins like azurin serve as electron mediators with their mono-nuclear copper site, the so-called type-1 copper site, as the active site. In type-1 copper sites the copper ion is strongly coordinated by three ligands, the S of a cysteine and the N’s of two histidines. Additionally, one or, in the case of azurin, possibly two, weaker axial ligands are present. The resulting trigonal pyramidal or distorted tetrahedral geometry is thought be intermediate between that preferred by Cu(I) and Cu(II) [Malmström, 1994; Williams, 1995]. This has been thought to make the type-1 copper sites ideally suited for electron transfer. In the used mutants specific features of the type-1 copper site are disrupted and it can be studied how the ET properties are affected. By applying modifications directly in the copper coordination sphere, another issue could be addressed. In order to understand better the role of the polypeptide matrix separating D and A, relevant ET routes have been identified using the structure-dependent pathway model developed by Beratan and Onuchic [Regan et al., 1993; Skourtis et al., 1994]. Two pathways have been recognised in the past which are about equally effective. In the present