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-methylglutaryl-CoA (HMG-CoA) lyase has been investigated by multifrequency electron spin resonance spectroscopy. Methodology has been developed to introduce copper in vitro into the isolated apoenzyme. The X-band EPR of Cu2+ (mixed isotopes or 63Cu2+) introduced in this way is very similar to the EPR spectra of samples in which copper is introduced during protein expression. g parallel and A parallel values in the X-band spectra support the prediction of nitrogen ligands to the tightly bound copper. In the g parallel region of S-band EPR spectra (mI = -1/2) of lyase-bound 63Cu2+, superhyperfine interactions due to nitrogen ligands are observed. Computer simulation with appropriate g values and A values and strain parameters was used to satisfactorily model both the X-band and the S-band spectra. The g parallel value of 2.282 and the A parallel value of 470 MHz are consistent with two nitrogen and two oxygen donor atoms for a square planar type 2 copper center. By simulating the mI = -1/2 line of the S-band spectrum, the superhyperfine features could be well modeled. This simulation approach was also used to distinguish between two and three nitrogen donor atoms. Based on the intensity patterns of the superhyperfine lines and the estimated coupling constants, it is concluded that at least two (and probably only two) nitrogen donor atoms are liganded to the tightly bound copper in HMG-CoA lyase. Additionally, kinetic experiments demonstrate that a spin-labeled substrate analog (R.CoA) is a competitive inhibitor of HMG-CoA lyase (KI = 98 microM). ESR titration experiments indicate that R.CoA binds to lyase with an equilibrium dissociation constant of 103 microM. Bound spin label exhibits a rotational correlation time, tau c, of 20 ns, in agreement with the value predicted for immobilization on a protein composed of two 32-kDa subunits.