Structural basis for VO(2+)-inhibition of nitrogenase activity: (B) pH-sensitive inner-sphere rearrangements in the 1H-environment of the metal coordination site of the nitrogenase Fe-protein identified by ENDOR spectroscopy.
The nitrogenase Fe-protein is the specific ATP-activated electron donor to the active site-containing nitrogenase MoFe-protein. It has been previously demonstrated that different VO(2+)-nucleotide coordination environments exist for the Fe-protein that depend on pH and are distinguishable by EPR spectroscopy. After having studied the nitrogenase 31P and 23Na superhyperfine structure for this system by electron nuclear double resonance (ENDOR) spectroscopy (Petersen et al. 2008 in J Biol Inorg Chem. doi:10.1007/s00775-008-0360-0), we here report on the 1H-interactions with the nucleotide-bound metal center after substitution of the natural diamagnetic metal Mg2+ with paramagnetic oxo-vanadium(IV). ENDOR spectra show a number of resonances arising from interactions of the VO2+ ion with protons. In the presence of reduced Fe-protein and VO2+ ADP, at least three sets of nonexchangeable protons are detected. At low pH the superhyperfine couplings of most of these are consistent with proton interactions originating from the nucleotide. There is no indication of 1H-resonances that exchange in D2O at neutral pH and could be assigned to inner-sphere hydroxyl coordination. Exchangeable hydroxyl protons in the inner coordination sphere with reduced Fe-protein are only found in the low pH form; based on their hyperfine tensor components these have been assigned to an axially coordinated hydroxyl water molecule. The pH-dependent alterations of the proton couplings that exchange in D2O suggest that they are partially caused by a rearrangement in the local hydroxyl coordination environment of the metal center. These rearrangements especially affect the apical metal position, where an axially coordinated water present at low pH is absent at neutral pH. Oxidation of the Fe-protein induced substantial changes in the electron-nucleus interactions. This indicates that the oxidation state of the iron-sulfur cluster has an important effect on the metal coordination environment at the nucleotide binding site of the Fe-protein. The distinct VO(2+)-nucleotide coordination structures with ADP and ATP and the redox state of the [4Fe-4S] cluster imply that VO2+ has a critical influence on the switch regions of the regulatory protein, and, taken together, this provides a plausible explanation for the inhibitory action of VO2+.
Petersen
Jan
J
Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. jan.petersen@bbsrc.ac.uk
Mitchell
Claire J
CJ
Fisher
Karl
K
Lowe
David J
DJ
D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007711:Klebsiella pneumoniae / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011522:Protons; D013379:Substrate Specificity; D017968:Vanadium Compounds / Q000737:chemistry* / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D011522:Protons; D017968:Vanadium Compounds; D010088:Oxidoreductases; C029130:nitrogenase reductase