Role of the Axial Ligand in Type 1 Cu Centers Studied by Point Mutations of Met148 in Rusticyanin

Abstract

Type 1 Cu centers in cupredoxins, nitrite reductases, and multi-copper oxidases utilize the same trigonal core ligation to His-Cys-His, with a weak axial ligand generally provided by a Met sulfur. In azurin, an additional axial ligand, a carbonyl oxygen from a Gly, is present. The importance of these axial ligands and in particular the Met has been debated extensively in terms of their role in fine-tuning the redox potential, spectroscopic properties, and rack-induced or entatic state properties of the copper sites. Extensive site-directed mutagenesis of the Met ligand has been carried out in azurin, but the presence of an additional carbonyl oxygen axial ligand has made it difficult to interpret the effects of these substitutions. Here, the axial methionine ligand (Met148) in rusticyanin is replaced with Leu, Gln, Lys, and Glu to examine the effect on the redox potential, acid stability, and copper site geometry. The midpoint redox potential varies from 363 (Met148Lys) to 798 mV (Met148Leu). The acid stability of the oxidized proteins is reduced except for the Met148Gln mutant. The Gln mutant remains blue at all pH values between 2.8 and 8, and has a redox potential of 563 mV at pH 3.2. The optical and rhombic EPR properties of this mutant closely resemble those of stellacyanin, which has the lowest redox potential among single-type 1 copper proteins (185 mV). The Met148Lys mutant exhibits type 2 Cu EPR and optical spectra in this pH range. The Met148Glu mutant exhibits a type 2 Cu EPR spectrum above pH 3 and a mixture of type 1 and type 2 Cu spectra at lower pH. The Met148Leu mutant exhibits the highest redox potential ( approximately 800 mV at pH 3.2) which is similar to the values in fungal laccase and in the type 1 Cu site of ceruloplasmin where this axial ligand is also a Leu.

Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations

“…Finally, the Met121Leu mutation also shifted the reduction potential ∼60-80 mV higher in each mutant (Table 2), which is consistent with reports by others and reasonable considering a stabilization of Cu(I) by the resulting trigonal ligand set [33,47,[85][86][87][88][89][90][91][92]. Overall, the Met121Leu triple Phe substituted mutant (Met121Leu + Leu33Phe/Met44Phe/ Leu86Phe azurin) showed the highest potential (503 mV vs. NHE), with a value over 140 mV higher than WT azurin.…”

High Resolution Structural Studies of Mutants Provide Insights into Catalysis and Electron Transfer Processes in Copper Nitrite Reductase

“…The 96 mV increase in redox potential in M144L is consistent with that observed in the equivalent M148L mutation in rusticyanin, where an increase of w130 mV was observed. 11 Indeed, the T1Cu sites of M144L AxNiR and M148L rusticyanin 12 are similar in structure. The T1Cu site is also similar to those seen in laccase 13 and ceruloplasmin, 14 which have leucine as the axial ligand and high redox potentials, although in the case of ceruloplasmin Leu adopted a different rotamer such that the nearest atom was w3.7 Å from the Cu.…”

Effects of axial ligand mutation of the type I copper site in bilirubin oxidase on direct electron transfer-type bioelectrocatalytic reduction of dioxygen

“…Site-directed point mutation of the axial ligand has been carried out for blue copper proteins. It has been reported that the replacement of Met at the axial ligand by Gln causes the negative shift in the formal potential of the type I Cu site in blue copper proteins [7][8][9][10][11]. In contrast, the replacement of Gln by Met causes the positive shift in the formal potential [12,13].…”