The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the co(II) derivative and co-EPR spectroscopy

Abstract

The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 A resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S(delta) atom is increased to 3.3-3.5 A and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1-2.4 A. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values gx' = 5.23; gy' = 3.83; gz' = 1.995, and hyperfine splittings Ax' = 31; Ay' = 20-30; Az' = 53 G) and indicates that the ligand field is close to axial.

A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774

“…However, the AK spectra are similar to those four-coordinated tetrahedral Co 2+ -complexes, as observed in model compounds, Co 2+ -reconstituted enzymes [46][47][48][49][50] and adenosine-triphosphate sulfurylases from D. desulfuricans and D. gigas [51]. Cobalt hyperfine splitting arising from the 59 Co nuclear spin (I = 7/2) has also been observed in some Co 2+ -substituted and Co 2+ -containing proteins [52,53]. Minor features at around g % 4.3 and g % 2.05 originate from adventitious high-spin iron(III) and copper(II) cavity contaminant, respectively.…”

Multifunctional 4-bit biomemory chip consisting of recombinant azurin variants

“…The results of metal-substituted azurin are shown in Fig. 2a,b,c,d, respectively [22,23]. Based on this analysis, the azurin variants shown to be well produced and could bind these metals (Fig.…”

“…The azurin variants were prepared by adding metal ions [0.5 M CoCl 2 , 0.5 M NiCl 2 ,6H 2 O, 0.5 M FeCl 2 ,nH2O, 0.5 M MnSO 4 ] to the apoazurin solution. Metal uptake of apoazurin required 3 days at 4 C. The metal substitutes azurins were purified by MWCO 5k Amicon Ultra centrifugal filter (Millipore, USA) [21,22].…”

Recent advances in understanding blue copper proteins

“…Here, we use this to further understand how metal-thiolate bonding changes with the nature of the metal ion through a study on a series of metal-varied model complexes with structures and spectra similar to metal substituted BC proteins [46][47][48][49][50][51][52][53][54][55]. These crystallographically characterized complexes of Prof. K. Fujisawa are of the general formula [ML(SC 6 F 5 )] where L = HB(3,5-i Pr 2 pz) 3 − (pyrazolyl (pz)) and M 2+ = Mn, Fe, Co, Ni, Cu,…”