Mn2+-adenosine nucleotide complexes in the presence of the nitrogenase iron-protein:: detection of conformational rearrangements directly at the nucleotide binding site by EPR and 2D-ESEEM (two-dimensional electron spin-echo envelope modulation spectroscopy)

Both ATP and a bivalent nucleotide-bound metal activator, normally Mg2+, are required for nitrogenase activity. EPR and ESEEM (electron spin-echo envelope modulation) measurements have been carried out on adenosine nucleotides in which the Mg2+ ion that is usually bound is replaced by Mn2+ in the presence of Kp2 (nitrogenase Fe-protein from Klebsiella pneumoniae). The Mn2+ zero-field splitting parameters have been determined from the EPR-spectrum to be vertical bar D vertical bar = 0.0125 cm(-1) with a rhombicity lambda = E/D = 0.31 by direct diagonalization of the complete spin Hamiltonian. ESEEM spectra of the Fe-protein with MnADP and MnATP both show an ESEEM line pair with one signal component at about 3.6 MHz and a relatively broad resonance at 8 MHz originating from a superhyperfine coupling to a P-31 nuclear spin from one or more directly co-ordinated phospho group(s) of the nucleotide. A pronounced resonance overlapping the low-frequency component of the P-31-signal at about 3.5 MHz is attributed to an interaction of Mn2+ with univalent Na-23 nuclei. ESEEM lines at frequencies < 3.5 MHz have been ascribed to interactions with N-14 nuclei. Differences in the N-14 features that depend on the type of nucleotide are consistent with substantial conformational rearrangements at the nucleotide-binding site upon hydrolysis. In addition, four-pulse HYSCORE (hyperfine sublevel correlation spectroscopy) experiments not only confirm the three-pulse ESEEM results, but also achieve significantly better spectral deconvolution, especially of the P-31-couplings, and demonstrate that the nucleotide is at least a unidentate ligand of Mn2+. Moreover it was also possible to identify peaks from an N-14 interaction more clearly; these most probably arise from outer-sphere interactions with nitrogen atom(s) of non-co-ordinated residues which are affected by conformational rearrangements upon nucleotide hydrolysis. In addition, different redox states of the [4Fe-4S] cluster of the Fe-protein show disparate conformations of the metal-nucleotide co-ordination environment, demonstrating that also the cluster site communicates with the nucleotide binding site.