Electron nuclear double resonance study of the Mn2+ environs in the oxalate-ATP complex of pyruvate kinase.

Abstract

Electron nuclear double resonance (ENDOR) and the related pulse technique of pulse field sweep EPR (PFSEPR) were used to probe the site I environment of Mn2+ in the oxalate-ATP complex of pyruvate kinase. Assignment of features and an estimate of hyperfine couplings have shown proximity of protons to the metal ions through their dipolar interaction and proximity of 31P and 17O because of a contact interaction from direct Mn(2+)-ligand covalent spin transfer. Since Mn2+ is a spin5/2 ion whose Ms = +/- 1/2, +/- 3/2, and +/- 5/2 electron spin states can all contribute to EPR and ENDOR, we have developed experimental and theoretical strategies for elucidating hyperfine couplings to the Mn2+ electron spin states. Solvent-exchangeable proton ENDOR features were evident with couplings very similar to the hyperfine couplings of H2O in [Mn(H2O)6]2+. ENDOR of exchangeable, more distant protons originated from a dipolar coupling such as could be expected from protons residing 5.5 A from Mn2+ and hydrogen-bonded to a nonliganding oxygen or nitrogen. Nonexchangeable proton ENDOR features indicated dipolar coupling to proton(s) from the protein residing at approximately 4.5 A from the Mn2+. The approximately 4-MHz 31P phosphate hyperfine couplings in Mn(II)-nucleotide models and in pyruvate kinase were similar, but a detailed ENDOR and PFSEPR comparison revealed that the hyperfine coupling to the ATP gamma-phosphate in pyruvate kinase was approximately 10% less than coupling to phosphates of Mn(II)-nucleotides. [In pyruvate kinase only the gamma-phosphate has been shown to bind to Mn2+ at site I (Lodato & Reed, 1987).](ABSTRACT TRUNCATED AT 250 WORDS)