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)

Abstract

Both ATP and a bivalent nucleotide-bound metal activator, normally Mg 2+ , are required for nitrogenase activity. EPR and ESEEM (electron spin-echo envelope modulation) measurements have been carried out on adenosine nucleotides in which the Mg 2+ ion that is usually bound is replaced by Mn 2+ in the presence of Kp2 (nitrogenase Fe-protein from Klebsiella pneumoniae). The Mn 2+ zero-field splitting parameters have been determined from the EPR-spectrum to be |D| = 0.0125 cm −1 with a rhombicity λ = 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 31 P nuclear spin from one or more directly co-ordinated phospho group(s) of the nucleotide. A pronounced resonance overlapping the lowfrequency component of the 31 P-signal at about 3.5 MHz is attributed to an interaction of Mn 2+ with univalent 23 Na nuclei. ESEEM lines at frequencies < 3.5 MHz have been ascribed to interactions with 14 N nuclei. Differences in the 14 N 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 31 P-couplings, and demonstrate that the nucleotide is at least a unidentate ligand of Mn 2+ . Moreover it was also possible to identify peaks from an 14 N interaction more clearly; these most probably arise from outersphere 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.

Insertion of heterometals into the NifEN-associated iron–molybdenum cofactor precursor

“…Our observation that V and Fe can be incorporated into the NifEN-associated precursor suggests the ability of Fe protein to mobilize metals other than Mo. Indeed, it was reported earlier that V and Mn can bind to the Fe protein in the presence of nucleotide [ 17 , 18 ]. Additionally, in the case of V, it has been demonstrated that V(V) (supplied in the form of VO 4 3− ) is reduced to V(IV) by the Fe protein before it can bind to the nucleotide binding site of the latter [ 17 ].…”

“…Indeed, it was reported earlier that V and Mn can bind to the Fe protein in the presence of nucleotide [17, 18]. Additionally, in the case of V, it has been demonstrated that V(V) (supplied in the form of VO 4 3− ) is reduced to V(IV) by the Fe protein before it can bind to the nucleotide binding site of the latter [17].…”

Mn2+–Nucleotide Coordination at the Myosin Active Site As Detected by Pulsed Electron Paramagnetic Resonance

“…A similar approach to determine the number of nearby magnetic nuclei ( 1 H, 13 C, 17 O) was previously used for Gd 3+ complexes. 12,37,38 The dramatic differences in the cfi of different Gd 3+ complexes in those works have necessitated a somewhat more elaborate procedure based on obtaining the separate ENDOR spectra for the |−1/2⟩ ↔ |1/2⟩ transition of the Gd 3+ spin S = 7/2 and then normalizing these spectra by the ESE amplitude associated with this transition.…”

Formation of Superoxo Species by Interaction of O2 with Na Atoms Deposited on MgO Powders: A Combined Continuous‐Wave EPR (CW‐EPR), Hyperfine Sublevel Correlation (HYSCORE) and DFT Study

“…The values fall within the typical range reported for this nucleus. [27,28] The absence of combination peaks appears to indicate that these interactions result from different superoxide species localised at different surface sites (speciation). This is consistent with the two different g zz factors (2.14 and 2.091) observed in the CW-EPR spectrum.…”