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 |D|=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 31P 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 (31)P-signal at about 3.5 MHz is attributed to an interaction of Mn2+ with univalent 23Na nuclei. ESEEM lines at frequencies <3.5 MHz have been ascribed to interactions with 14N nuclei. Differences in the 14N 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 31P-couplings, and demonstrate that the nucleotide is at least a unidentate ligand of Mn2+. Moreover it was also possible to identify peaks from an 14N 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.
Petersen
Jan
J
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK. jan.petersen@bbsrc.ac.uk
Gessner
Christof
C
Fisher
Karl
K
Mitchell
Claire J
CJ
Lowe
David J
DJ
Lubitz
Wolfgang
W
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008345:Manganese / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods*
D008345:Manganese; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C029130:nitrogenase reductase