The pulsed EPR methods of electron spin echo envelope modulation (ESEEM) and electron spin echo–electron nuclear double resonance (ESE–ENDOR) are used to investigate the proximity of exchangeable hydrogens around the paramagnetic S
2
–state Mn cluster of the photosystem II oxygen–evolving complex. Although ESEEM and ESE–ENDOR are both pulsed electron paramagnetic resonance techniques, the specific mechanisms by which nuclear spin transitions are observed are quite different. We are able to generate good simulations of both
1
H ESE–ENDOR and
2
H ESEEM signatures of exchangeable hydrogens at the S
2
–state cluster. The convergence of simulation parameters for both methods provides a high degree of confidence in the simulations. Several exchangeable protons–deuterons with strong dipolar couplings are observed. In the simulations, two of the close (≈2.5 Å) hydrogen nuclei exhibit strong isotropic couplings and are therefore most probably associated with direct substrate ligation to paramagnetic Mn. Another two of the close (≈2.7 Å) hydrogen nuclei show no isotropic couplings and are therefore most probably not contained in Mn ligands. We suggest that these proximal hydrogens may be associated with a Ca
2+
–bound substrate, as indicated in recent mechanistic proposals for O
2
formation.
Pulsed Electron-Electron Double Resonance (PELDOR) and Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy in Bioanalysis
