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. 2016 Oct 4;113(40):E5783-E5791.
doi: 10.1073/pnas.1613089113.

Negative Cooperativity in the Nitrogenase Fe Protein Electron Delivery Cycle

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Free PMC article

Negative Cooperativity in the Nitrogenase Fe Protein Electron Delivery Cycle

Karamatullah Danyal et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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.

Keywords: ATP hydrolysis; allosteric control; conformational control; half-sites reactivity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Nitrogenase complex. (A) Ribbon diagram of the symmetrical ternary [MoFe(Fered (ATP)2)2] complex (PDB ID code 1G21) with the MoFe protein subunits in salmon (α subunits) and blue (β subunits) at the center with two Fe proteins (green) bound on each end. Distances between the ATP binding sites on one Fe protein and the P cluster, the interface on the opposite face, and to the ATP binding sites on the other Fe protein are noted. (B) A cartoon schematic of nitrogenase is shown with the [4Fe-4S] cluster of the Fe protein as red cubes in both Fe proteins, the P clusters as white rectangles, and FeMo-co as white diamonds. The path of electron movement is denoted by arrows and the numbers (1) and (2) and indicate the order of ET.
Fig. 2.
Fig. 2.
Presteady-state ATP hydrolysis. Presteady-state, quench-flow measurements of the time course of ATP hydrolysis by nitrogenase (micromoles of ADP formed per micromole of MoFe protein). The reaction was initiated by mixing MoFe protein (10 μM) against Fe protein (40 μM) with [α-32P]ATP (2 mM) with quenching at the noted times by addition of acetic acid. ADP formed (●) was quantified as noted in Materials and Methods. A shows the time course up to 500 ms, and B shows the time course up to 100 ms. The solid lines are a fit to a burst phase followed by a linear steady state (Eq. 3), where kATP = 38 s−1, A = 1.96 μM ADP/μM MoFe protein, and V = 11.8 μM ADP (μM MoFe)−1⋅s−1.
Fig. 3.
Fig. 3.
ET kinetics. Presteady-state, SF measurements of the absorbance change at 430 nm (left ordinate) for ET from Fered to MoFe protein within the [MoFe(Fered(ATP)2)2] complex. Red line, result of global fit to negative cooperativity in Fig. 4, Scheme C, using the derived rate constants listed; right ordinate, stoichiometry of reduction derived from Scheme C. Conditions: MoFe protein (10 µM) is mixed with Fe protein (37.5 µM) in a ratio of ∼1:4 with 10 mM MgATP in an SF spectrophotometer.
Fig. 4.
Fig. 4.
Kinetic schemes. (Scheme A) Independent-sites model: Each half of the [MoFe(Fered(ATP)2)2] complex functions independently. (Scheme B) Half-sites reactivity model: One-half of the complex undergoes electron delivery, and in so doing completely suppresses electron delivery in the other half. (Scheme C) Negative cooperativity model: rate constants listed on the scheme were obtained by freely floating these parameters in the global fit to the differential equations of the scheme (SI Appendix) as described in the text; parentheses contain the statistical 95% confidence limits (±). Also as described, redox state of the s and p halves of MoFe protein are not indicated because electron delivery is independent of MoFe protein reduction level. Recharging (bottom): Simplified scheme in which Fered(ATP)2 is regenerated by rapid reduction of Feox and ATP binding and MoFe protein binding Fered(ATP)2 in a single second-order step for binding of Fered(ATP)2 to MoFe at the reported second-order rate constant, k1 = 5 × 107 M−1⋅s−1 (19), because the overall process is almost instantaneous relative to the other reactions in the scheme.
Fig. 5.
Fig. 5.
ATP hydrolysis data fits. Presteady-state, quench-flow measurements of the time course of ATP hydrolysis by rapidly mixing MoFe protein (10 μM) and Fe protein (40 μM) with [α-32P]ATP (2 mM) (●). Dashed black line: half-sites model, Scheme B, using rate constants, kET = 140 s−1, kATP = 36 s−1, kPi = 16 s−1, and koff = 11.9 s−1, as derived from the phenomenological fits to the experimental data, along with the recharging model. Solid black line: independent-sites model, Scheme A, calculated analogously. Red line: calculated from the rate parameters obtained by the global fit to negative cooperativity Scheme C, as given in the scheme. (Inset) Data and simulations to longer times.
Fig. 6.
Fig. 6.
Kinetic schemes with negative cooperativity manifest at tiers 1 and 2. Simplified negative cooperativity schemes with branching between primary (p) and seconday (s) branches at tiers 1 and 2. As a notable simplification discussed in the text, along the p branch the complex exhibits half-sites reactivity. When ET occurs in the second half this generates the s branch where the two halves of the complex react. When this branching ET occurs at tier 1 in competition with ATP hydrolysis, the two halves of the resulting symmetrical s branch complex react synchronously in subsequent steps along the reaction pathway. When ET in the second half occurs at tier 2, in competition with Pi release, the second half undergoes prompt ATP hydrolysis to again create a symmetrical s-branch complex, and the two halves subsequently react synchronously. Tier 3 follows straightforwardly.
Fig. 7.
Fig. 7.
Correlated movement of the two Fe proteins. (A) Covariance matrix (in square angstroms) showing the correlation between the displacement of amino acid residues in ATP-bound Fe protein nitrogenase complex. For sake of clarity, the matrix is divided into quadrants corresponding to the various possible subunit pairs (see Fig. 1). Regions of correlated (in-phase) and anticorrelated (out-of-phase) motions are shown in red and blue, respectively. The regions of cross-correlation between the two Fe proteins (upper right corner) are highlighted with dotted boxes. (B) Collective motion corresponding to the rocking motion of the Fe protein on the MoFe protein surface is depicted. The position of the Fe proteins (green) in the ADP-bound structure (PDB ID code 2AFI) is shown using a faded cartoon representation. The length of the arrows is proportional to the displacement of the amino acid residues obtained through coarse-grained normal mode analysis.

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