Blue Copper Proteins

Cd- and Hg-Derivatives of Blue Copper Proteins: Coordination and Rigidity of Metal Sites

B. Cortecka, C. Lippert, X. Ni, W. Tröger, T. Butz

Nuclear Solid State Physics, University of Leipzig

Cd- and Hg-derivatives of the multi-copper enzymes ascorbate oxidase and laccase as well as the small blue copper proteins (electron transfer proteins) azurin, plastocyanin, and stellacyanin are prepared with the shortlived radioisotopes 111mCd (t1/2=49 min) and 199mHg (t1/2=43 min) supplied by the isotope separator ISOLDE.  The nuclear quadrupole interaction (NQI) of 111mCd and 199mHg in these proteins is monitored by time differential perturbed angular correlation  of g-rays (TDPAC).

In the Hg-derivatives of the multi-copper proteins three sites have been detected  and assigned to the so-called type-1, type-2, and type-3 metal sites. Our results  on the Cd-derivatives show that Cd does not reconstitute the type-2 site at all.

The type-1 site is clearly identified by comparing the NQI data with those of small  blue copper proteins containing the type-1 site only.
The type-1 site in the native  proteins azurin, plastocyanin, and stellacyanin as well as various mutants of azurin  and plastocyanin has been studied with both 111mCd- and 199mHg- TDPAC.
The type-1 metal coordination appears to be mainly three-fold planar by one cysteine and two histidines (bond length about 2 Angstrom). A longer approach is made  by a methionine perpendicular to the plane: bond length about 3 Angstrom . In  order to assess its role in the metal coordination, the methionine was exchanged  against other ligands via site directed mutagenesis. This has a small effect on the  NQI only which is easily understood in terms of a small metal displacement out of the plane. This is a clear indication that the methionine does not bind to the metal but may have some importance for the fine tuning of this "entatic" metal coordination.

Furthermore, the dynamic properties of the metal sites in these system are investigated by 111mCd-TDPAC.

The electric field gradient tensor components of the 111mCd/199mHg -NQI's in type-1 and type-3 sites of the investigated proteins appear to scale for the 111mCd- and 199mHg-derivatives indicating a torsional rigidity of the metal site.

 

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