Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

Significance Nitrogenase catalyzes N2 reduction to ammonia, the largest N input into the biogeochemical nitrogen cycle. This difficult reaction involves delivery of electrons from the Fe protein component to the catalytic MoFe protein component in a process that involves hydrolysis of two ATP per electron delivered. MoFe contains two catalytic halves, each of which binds an Fe protein. The prevailing picture has been that the two halves function independently. Here, it is demonstrated that electron transfer (ET) in the two halves exhibits negative cooperativity: Fe→MoFe ET in one-half partially suppresses ET in the other. These findings thus show that conformational coupling in nitrogenase not only gates ET within each half, as shown previously, but introduces negative cooperativity between the two halves. Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.

[1]  J. W. Peters,et al.  Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle. , 2016, Biochemistry.

[2]  D. Rees,et al.  Structural Evidence for Asymmetrical Nucleotide Interactions in Nitrogenase , 2014, Journal of the American Chemical Society.

[3]  L. Seefeldt,et al.  A Confirmation of the Quench-Cryoannealing Relaxation Protocol for Identifying Reduction States of Freeze-Trapped Nitrogenase Intermediates , 2014, Inorganic chemistry.

[4]  L. Seefeldt,et al.  Substrate channel in nitrogenase revealed by a molecular dynamics approach. , 2014, Biochemistry.

[5]  Dennis R. Dean,et al.  Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage , 2014, Chemical reviews.

[6]  L. Seefeldt,et al.  Electron transfer precedes ATP hydrolysis during nitrogenase catalysis , 2013, Proceedings of the National Academy of Sciences.

[7]  L. Seefeldt,et al.  Nitrogenase: a draft mechanism. , 2013, Accounts of chemical research.

[8]  L. Seefeldt,et al.  Electron transfer in nitrogenase catalysis. , 2012, Current opinion in chemical biology.

[9]  D. Rees,et al.  Evidence for Interstitial Carbon in Nitrogenase FeMo Cofactor , 2011, Science.

[10]  L. Seefeldt,et al.  Electron transfer within nitrogenase: evidence for a deficit-spending mechanism. , 2011, Biochemistry.

[11]  S. Fairhurst,et al.  Electron transfer and half-reactivity in nitrogenase. , 2011, Biochemical Society transactions.

[12]  L. Seefeldt,et al.  Conformational gating of electron transfer from the nitrogenase Fe protein to MoFe protein. , 2010, Journal of the American Chemical Society.

[13]  K. Hodgson,et al.  Stepwise formation of P-cluster in nitrogenase MoFe protein , 2009, Proceedings of the National Academy of Sciences.

[14]  K. Fisher,et al.  Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function. , 2007, Journal of inorganic biochemistry.

[15]  Debarshi Mustafi,et al.  Nitrogenase Complexes: Multiple Docking Sites for a Nucleotide Switch Protein , 2005, Science.

[16]  Lance C Seefeldt,et al.  Nitrogen Fixation: The Mechanism of the Mo-Dependent Nitrogenase , 2003, Critical reviews in biochemistry and molecular biology.

[17]  D. Rees,et al.  Nitrogenase MoFe-Protein at 1.16 Å Resolution: A Central Ligand in the FeMo-Cofactor , 2002, Science.

[18]  P. E. Wilson,et al.  Duplication and extension of the Thorneley and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using a MATHEMATICA software platform. , 2001, Biophysical chemistry.

[19]  S. Fairhurst,et al.  Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase , 2001, JBIC Journal of Biological Inorganic Chemistry.

[20]  K. Fisher,et al.  Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover: evidence for S = 3/2 spin states from reduced MoFe-protein intermediates. , 2001, Biochemistry.

[21]  J. W. Peters,et al.  MgATP-Bound and nucleotide-free structures of a nitrogenase protein complex between the Leu 127 Delta-Fe-protein and the MoFe-protein. , 2001, Biochemistry.

[22]  R. Jernigan,et al.  Anisotropy of fluctuation dynamics of proteins with an elastic network model. , 2001, Biophysical journal.

[23]  R. Stroud,et al.  The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits. , 1999, Biochemistry.

[24]  W. Lanzilotta,et al.  Thermodynamics of nucleotide interactions with the Azotobacter vinelandii nitrogenase iron protein. , 1999, Biochimica et biophysica acta.

[25]  W. Lanzilotta,et al.  Catalytic and biophysical properties of a nitrogenase Apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii. , 1998, Biochemistry.

[26]  D. Rees,et al.  Structure of ADP·AIF4 –-stabilized nitrogenase complex and its implications for signal transduction , 1997, Nature.

[27]  B. Burgess,et al.  Mechanism of Molybdenum Nitrogenase. , 1996, Chemical reviews.

[28]  Tirion,et al.  Large Amplitude Elastic Motions in Proteins from a Single-Parameter, Atomic Analysis. , 1996, Physical review letters.

[29]  M. Webb,et al.  ATP Hydrolysis and Energy Transduction by Nitrogenase , 1995 .

[30]  D. Rees,et al.  Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. , 1992, Science.

[31]  J. Howard,et al.  Effect of salts on Azotobacter vinelandii nitrogenase activities. Inhibition of iron chelation and substrate reduction. , 1990, The Journal of biological chemistry.

[32]  G. Watt,et al.  Redox reactions of and nucleotide binding to the iron protein of Azotobacter vinelandii , 1986 .

[33]  R. Thorneley,et al.  The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution. , 1984, The Biochemical journal.

[34]  G. L. Anderson,et al.  Reactions with the oxidized iron protein of Azotobacter vinelandii nitrogenase: formation of a 2Fe center. , 1984, Biochemistry.

[35]  R. Thorneley,et al.  Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction. , 1983, The Biochemical journal.

[36]  J. Dormand,et al.  A family of embedded Runge-Kutta formulae , 1980 .

[37]  R. Burris,et al.  Nitrogenase: the reaction between the Fe protein and bathophenanthrolinedisulfonate as a probe for interactions with MgATP. , 1978, Biochemistry.

[38]  J. Hopfield.  On electron transfer. , 1976, Biophysical journal.

[39]  R. Thorneley.  Nitrogenase of Klebsiella pneumoniae. A stopped-flow study of magnesium-adenosine triphosphate-induce electron transfer between the compeonent proteins. , 1975, The Biochemical journal.

[40]  R. Hardy,et al.  Nitrogenase: The Catalysis , 1975 .

[41]  D. Koshland,et al.  Half-of-the sites reactivity and negative co-operativity: the case of yeast glyceraldehyde 3-phosphate dehydrogenase. , 1973, Journal of molecular biology.

[42]  D. Koshland,et al.  Half-of-the-sites reactivity and the conformational states of cytidine triphosphate synthetase. , 1971, Biochemistry.

[43]  D. Koshland,et al.  Negative cooperativity in enzyme action. The binding of diphosphopyridine nucleotide to glyceraldehyde 3-phosphate dehydrogenase. , 1968, Biochemistry.