REACTIVITY OF PSEUDOAZURIN FROM ACHROMOBACTER-CYCLOCLASTES WITH INORGANIC REDOX PARTNERS AND RELATED NMR AND ELECTROCHEMICAL STUDIES

The effect of pH on rate constants (25-degrees-C) for the [Co(dipic)2] - and [Co(phen)3]3+ oxidations of pseudoazurin pACu(I) have been studi ed in the pH range 3.5-8.7. From the trends observed (decrease in rate constants with decreasing pH) two pK(a)'s are obtained, and from H-1 NMR these are confirmed as being associated with an active site His pr otonation/deprotonation, pK(a) = 4.84 (average), and a protonation/dep rotonation of the uncoordinated His6, pK(a) = 7.21 (average). Alongsid e plastocyanin and amicyanin, pseudoazurin therefore provides a third example of a type 1 protein the Cu(I) state of which exhibits an activ e site protonation in the accessible pH range. The spacings of the act ive site His, Cys, and Met coordinated residues are noted and appear t o relate to the magnitude of the pK(a) values observed. The rate const ant for the oxidation with [Fe(CN)6]3- is >2.6 X 10(6) M-1 s-1 at pH 5 .5, yielding an exceptionally high k(Fe)/k(Co) ratio of >7.8 x 10(3) a t this pH, where k(Co) 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 discuss ed. The presence of these basic residues explains the relatively small value of 2.9 x 10(3) M-1 s-1 for the electron self-exchange rate cons tant at 25-degrees-C, I = 0.100 M.