Determination of active-site conformations in paramagnetic myoglobin from analyses of cross-relaxation and hyperfine shifts

Abstract

A methodology for determining the haem vinyl, haem propionate α-CH₂ and proximal His F8 C_α–C_β conformations in the active site of haemoproteins has been elaborated. It is based on measurements of interproton distances using the time-dependent nuclear Overhauser effect (NOE) and analysis of hyperfine shifts. The initial build-up slope of the NOE between selected protons for each fragment provides the cross-relaxation rate which can be interpreted in terms of the interproton distance. Secondary NOEs and spin diffusion are observed for the met-cyano form of myoglobin used. However, the primary NOE can be identified from the initial NOE build-up slope. Owing to the symmetric nature of the fragments, two conformational states are obtained for a given interproton distance. The analysis of the hyperfine shifts provides complementary structural information to that obtained from the NOE results. Consequently, specific conformations for these fragments can be uniquely determined from combined analyses of the cross-relaxation rates and hyperfine shifts.