Reactivity of Pseudoazurin from Achromobacter cycloclastes with Inorganic Redox Partners and Related NMR and Electrochemical Studies

Abstract

The effect of pH on rate constants (25 "C) for the [Co(dipic)2]-and [C0(phen)~]3+ oxidations of pseudoazurin pACu' have been studied in the pH range 3.5-8.7. From the trends observed (decrease in rate constants with decreasing pH) two pK. ' s are obtained, and from IH NMR these are confirmed as being associated with an active site His protonationfdeprotonation, pK, = 4.84 (average), and a protonationfdeprotonation of the uncoordinated H i d , pK, = 7.2 1 (average). Alongside plastocyanin and amicyanin, pseudoazurin therefore provides a third example of a type 1 protein the Cu(1) state of which exhibits an active site protonation in the accessible pH range. The spacings of the active site His, Cys, and Met coordinated residues are noted and appear to relate to the magnitude of the pK, values observed. The rate constant for the oxidation with [Fe(CN)@-is >2.6 X 106 M-l s-I at pH 5.5, yielding an exceptionally high k~, f k a ratio of >7.8 X lo3 at this pH, where ka is for the [Co(phen)3]3+ oxidation.The effect of a number of conserved basic residues Lys38, Lys46, Lys57, and Lys77 (the last adjacent to the active site Cys78) on reactivity is discussed. The presence of these basic residues explains the relatively small value of 2.9 X lo3 M-l s-I for the electron self-exchange rate constant at 25 OC, I = 0.100 M.

pH-dependent transition between delocalized and trapped valence states of a Cu A center and its possible role in proton-coupled electron transfer

“…In blue copper proteins such as WT azurin, the C-terminal His is also at a similar location (47). Protonation of the C-terminal His ligand has been shown to occur in the reduced state of selected blue copper proteins such as plastocyanin, amicyanin, and pseudoazurin (48)(49)(50)(51)(52). However, no evidence for protonation has been found in the reduced state of other blue copper proteins such as azurin (53).…”

Crystal Structure Determinations of Oxidized and Reduced Pseudoazurins from Achromobacter cycloclastes

“…The loss of a similar water molecule was found in A. faecalis pseudoazurin at both pH 7.8 and 7.0, while the peptide bond flip was found at only pH 7.0. Because the pK a values of the uncoordinated His 6 residue are 7.2 (reduce form) and 6.5 (oxidized form) (21), the protonation at His 6 may be the cause of the peptide bond flip in Al. faecalis pseudoazurin.…”

“…Recent kinetic and theoretical studies on the blue copper protein plastocyanin have indicated the presence of two distinct electron-transfer sites: (i) the adjacent hydrophobic patch ϳ6 Å from the copper through which one of the histidine ligands protrudes, and (ii) the remote site involving and acidic patch region ϳ15 Å from the copper, with acidic residues on either side of the exposed 3 ] 3ϩ oxidation (21), indicating a clear preference of the protein for anionic species. Moreover, the self-exchange rate constant for reduced molecule (about 3 ϫ 10 3 M Ϫ1 s Ϫ1 ) is much smaller than those of most other cupredoxins and is quite similar to that found in higher plant plas-tocyanins (22).…”

“…The effect of pH on the redox reactivity of A. cycloclastes pseudoazurin is well documented (21,22,45). The reduced protein has been shown to participate in an active site protonation/deprotonation equilibrium at His 81 , which gives an acid dissociation pK a of 5.2 from an NMR titration (21) and 4.6 from kinetic studies with inorganic complexes as oxidants (45).…”

“…The reduced protein has been shown to participate in an active site protonation/deprotonation equilibrium at His 81 , which gives an acid dissociation pK a of 5.2 from an NMR titration (21) and 4.6 from kinetic studies with inorganic complexes as oxidants (45). The effect of protonation/deprotonation of uncoordinated His 6 residue located near to the active site shows a pK a values of 7.2 (reduced form) and 6.5 (oxidized form) from NMR titration and 7.3 from kinetic studies of the oxidation of the reduced pseudoazurin with small complexes (21).…”