Structural basis for VO2+ inhibition of nitrogenase activity (A): 31P and 23Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy

Original Paper


We previously reported the vanadyl hyperfine couplings of VO2+–ATP and VO2+–ADP complexes in the presence of the nitrogenase Fe protein from Klebsiella pneumoniae (Petersen et al. in Biochemistry 41:13253–13263, 2002). It was demonstrated that different VO2+–nucleotide coordination environments coexist and are distinguishable by electron paramagnetic resonance (EPR) spectroscopy. Here orientation-selective continuous-wave electron–nuclear double resonance (ENDOR) spectra have been investigated especially in the low-radio-frequency range in order to identify superhyperfine interactions with nuclei other than protons. Some of these resonances have been attributed to the presence of a strong interaction with a 31P nucleus although no resolvable superhyperfine structure due to 31P or other nuclei was detected in the EPR spectra. The superhyperfine coupling component is determined to be about 25 MHz. Such a 31P coupling is consistent with an interaction of the metal with phosphorus from a directly, equatorially coordinated nucleotide phosphate group(s). Additionally, novel more prominent 31P ENDOR signals are detected in the low-frequency region. Some of these correspond to a relatively weak 31P coupling. This coupling is present with ATP for all pH forms but is absent with ADP. The ENDOR resonances of these weakly coupled 31P are likely to originate from an interaction of the metal with a nucleotide phosphate group of the nucleoside triphosphate and are attributed to a phosphorus with axial characteristics. Another set of resonances, split about the nuclear Zeeman frequency of 23Na, was detected, suggesting that a monovalent Na+ ion is closely associated with the divalent metal–nucleotide binding site. Na+ replacement by K+ unambiguously confirmed that ENDORs at radio frequencies between 3.0 and 4.5 MHz arise from an interaction with Na+ ions. In contrast to the low-frequency 31P signal, these resonances are present in spectra with both ADP and ATP, and for both low- and neutral-pH forms, although slight differences are detected, showing that these are sensitive to the nucleotide and pH.


Metalloenzymes Nitrogenase Fe protein Nucleotides Electron paramagnetic resonance 



Adenosine 5′-diphosphate


Adenosine 5′-triphosphate


Electron–nuclear double resonance


Electron paramagnetic resonance


Electron spin echo envelope modulation


Hyperfine sublevel correlation


Nitrogenase Fe protein from Klebsiella pneumoniae





We gratefully acknowledge the help of C.J. Mitchell for preparing some of the samples. EPR simulations of the VO spectra were conducted using the program LSIM written by D. Collison and kindly provided by S. Fairhurst. D.J.L. thanks BBSRC for financial support through the Core Strategic Grant to the John Innes Centre.

Supplementary material


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Copyright information

© SBIC 2008

Authors and Affiliations

  1. 1.Department of Biological ChemistryJohn Innes CentreNorwichUK
  2. 2.Manchester Interdisciplinary BiocentreThe University of ManchesterManchesterUK

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