Alkaline Transition of Pseudoazurin from Achromobacter cycloclastes Studied by Paramagnetic NMR and Its Effect on Electron Transfer

Abstract

Paramagnetic NMR studies on the Cu(II) form of pseudoazurin have been used to demonstrate that the alkaline transition of this protein results in an active site in which the axial Cu-S(Met) interaction is considerably decreased. This observation confirms the conclusion made from various other spectroscopic methods. Furthermore, we show that the alkaline transition of pseudoazurin coincides with a dramatic increase in the electron self-exchange rate constant of the protein. The self-exchange rate constant (25 °C) at pH 8.2 is 3.5 × 10 3 M -1 s -1 (I ) 0.10 M), consistent with a previously determined value (25 °C) of 2.9 × 10 3 M -1 s -1 (I ) 0.10 M) at pH 7.5. Upon increasing the pH value to 10.9 the self-exchange rate constant (25 °C) increases to 1.7 × 10 4 M -1 s -1 (I ) 0.10 M). The increased self-exchange reactivity at high pH is due to the deprotonation of a number of lysine residues that surround the hydrophobic patch of the protein, the most likely docking surface for the self-exchange process. The concomitant active site changes indicate that the deprotonation of one or more surface lysine residues is responsible for the alkaline transition in pseudoazurin.

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

“…PAz isolated from P. pantotrophus has a formal reduction potential of 230 ± 5 mV, at pH 7.0, and three pK values were identified, 6.5, 7.2 and 10.5, from the dependence of that parameter with the pH, as verified for other small blue copper proteins [22,33,34].…”

“…Some of the pK values can be assigned from spectroscopic evidence for this PAz [16], by comparison with the results obtained for PAz from Achromobacter cycloclastes [22,33,34], which has 67% homology with P. pantotrophus PAz. The pK values of 6.5 and 7.2 have been assigned to the protonation of the histidine residue 6 [33].…”

Active-Site Structure and Electron-Transfer Reactivity of Plastocyanins

“…The self-exchange process in PCu's (and all cupredoxins) is thought to involve the association of two protein molecules via the hydrophobic surfaces which surround the His87 ligand. 43,68,70,71,74 Given the comparable active-site structures of the Cu(II) proteins which we have demonstrated in the paramagnetic 1 H NMR studies and which, given the rigidity of the cupredoxin fold, also hold for the Cu-(I) forms (at a pH value well above the pK a of His87) we can anticipate that the reorganization energies of the various PCu's are similar. Additionally, we have shown that the spin density distributions onto the ligands are alike in all of the PCu(II)s, and thus variations in this feature will not influence et.…”

“…The pseudocontact shifts are small for protons at a cupric type 1 site, [41][42][43][44][45][46][47] due to the small anisotropy of the g tensor, and range from ∼2 to -3 ppm for the resonances listed in Table 1. 42,46 Therefore, the δ obs value minus the δ dia value (see Tables S1 and S2 in the Supporting Information for typical δ dia values) for a particular proton listed in Table 1 provide a good estimate of δ Fc , which is a measure of the spin density present at that particular proton.…”

“…The technique which we have chosen for this purpose is paramagnetic proton nuclear magnetic resonance ( 1 H NMR) spectroscopy as it has recently been shown to provide extremely detailed information about the active-site structure of Cu(II) cupredoxins, including the spin density distribution onto the ligands. [41][42][43][44][45][46][47] …”