P-MEVALONII 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE - ELECTRON-PARAMAGNETIC-RESONANCE INVESTIGATION OF THE COPPER-BINDING SITE

The copper binding site in Pseudomonas mevalonii 3-hydroxy-3-methylglu taryl-CoA (HMG-CoA) lyase has been investigated by multifrequency elec tron spin resonance spectroscopy. Methodology has been developed to in troduce copper in vitro into the isolated apoenzyme. The X-band EPR of Cu2+ (mixed isotopes or Cu-63(2+)) introduced in this way is very sim ilar to the EPR spectra of samples in which copper is introduced durin g protein expression. g(parallel to) and A(parallel to) values in the X-band spectra support the prediction of nitrogen ligands to the tight ly bound copper. In the g(parallel to) region of S-band EPR spectra (m (I) = -1/2) of lyase-bound Cu-63(2+), superhyperfine interactions due to nitrogen ligands are observed. Computer simulation with appropriate g values and A values and strain parameters was used to satisfactoril y model both the X-band and the S-band spectra. The g(parallel to) val ue of 2.282 and the A(parallel to) value of 470 MHz are consistent wit h two nitrogen and two oxygen donor atoms for a square planar type 2 c opper center. By simulating the m(I) = -1/2 line of the S-band spectru m, the superhyperfine features could be well modeled. This simulation approach was also used to distinguish between two and three nitrogen d onor atoms. Based on the intensity patterns of the superhyperfine line s and the estimated coupling constants, it is concluded that at least two (and probably only two) nitrogen donor atoms are liganded to the t ightly bound copper in HMG-CoA lyase. Additionally, kinetic experiment s demonstrate that a spin-labeled substrate analog (R(.)CoA) is a comp etitive inhibitor of HMG-CoA lyase (K-I = 98 mu M). ESR titration expe riments indicate that R(.)CoA binds to lyase with an equilibrium disso ciation constant of 103 mu M. Bound spin label exhibits a rotational c orrelation time, tau(c), of 20 ns, in agreement with the value predict ed for immobilization on a protein composed of two 32-kDa subunits. (C ) Academic Press, Inc.