ABSTRACT
Nitrogenase is the only enzyme that can cleave the triple bond in N2, making nitrogen available to plants (although the enzyme itself is strictly microbial). It has been studied extensively with both experimental and computational methods, but many details of the reaction mechanism are still unclear. X-ray crystallography is the main source of structural information for biomacromolecules, but it has problems to discern hydrogen atoms or to distinguish between elements with the same number of electrons. These problems can sometimes be alleviated by introducing quantum chemical calculations in the refinement, providing information about the ideal structure (in the same way as the empirical restraints used in standard crystallographic refinement) and comparing different interpretations of the structure with normal crystallographic and quantum mechanical quality measures. We have performed such quantum-refinement calculations to address two important issues for nitrogenase. First, we show that the bidentate ligand of the active-site FeV cluster in Vnitrogenase is carbonate, rather than bicarbonate or nitrate. Second, we study the CO-inhibited structure of Monitrogenase. CO binds to a reduced and protonated state of the enzyme by replacing one of the sulfide ions (S2B) in the active-site FeMo cluster. We examined if it is possible to deduce from the crystal structure the location of the protons. Our results indicates that the crystal structure is best modelled as fully deprotonated.
Subject(s)
Carbon Monoxide/chemistry , Iron/chemistry , Molybdenum/chemistry , Nitrogenase/chemistry , Carbonates/chemistry , Catalytic Domain , Crystallography, X-Ray/methods , Electrons , Ligands , Models, Molecular , Nitrogenase/ultrastructure , Protons , Quantum Theory , Sulfides/chemistryABSTRACT
Surface residues, interior residues, and parsing residues, together with a secondary structure derived from these, are predicted for the MoFe nitrogenase protein in advance of a crystal structure of the protein, scheduled shortly to appear in Nature. By publishing this prediction, we test our method for predicting the conformation of proteins from patterns in the divergent evolution of homologous protein sequences in a way that places the method 'at risk'.
Subject(s)
Nitrogenase/chemistry , Amino Acid Sequence , Azotobacter vinelandii/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/ultrastructure , Crystallography , Iron , Metalloproteins/chemistry , Metalloproteins/ultrastructure , Models, Theoretical , Molecular Sequence Data , Molybdenum , Nitrogenase/ultrastructure , Protein Structure, Secondary , Sequence AlignmentABSTRACT
A plausible structure of the iron-molybdenum cofactor of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1] is presented based on altered substrate reduction properties of dinitrogenase containing homocitrate analogs within the cofactor. Alterations on each carbon of the four-carbon homocitrate backbone were correlated with altered substrate reduction properties of dinitrogenase containing these analogs. Altered substrate reduction properties are the basis for a model in which homocitrate is oriented about two cubane metal clusters.
