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. 2018 Sep 18;51(9):2179-2186.
doi: 10.1021/acs.accounts.8b00112. Epub 2018 Aug 10.

Energy Transduction in Nitrogenase

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Free PMC article

Energy Transduction in Nitrogenase

Lance C Seefeldt et al. Acc Chem Res. .
Free PMC article

Abstract

Nitrogenase is a complicated two-component enzyme system that uses ATP binding and hydrolysis energy to achieve one of the most difficult chemical reactions in nature, the reduction of N2 to NH3. One component of the Mo-based nitrogenase system, Fe protein, delivers electrons one at a time to the second component, the catalytic MoFe protein. This process occurs through a series of synchronized events collectively called the "Fe protein cycle". Elucidating details of the events associated with this cycle has constituted an important challenge in understanding the nitrogenase mechanism. Electron delivery is a multistep process involving three metal clusters with intra- and interprotein events. It is proposed that the first electron transfer event is a gated intraprotein transfer of one electron from the MoFe protein P-cluster to the FeMo cofactor. Measurement of the effect of osmotic pressure on the rate of this electron transfer process revealed that it is gated by protein conformational changes. This first electron transfer is activated by binding of the Fe protein containing two bound ATP molecules. The mechanism of how this protein-protein association triggers electron transfer remains unknown. The second electron transfer event is proposed to be a rapid interprotein "backfill" with transfer of one electron from the reduced Fe protein 4Fe-4S cluster to the oxidized P-cluster. In this way, electron delivery can be viewed as a case of "deficit spending". Such a deficit-spending electron transfer process can be envisioned as a way to achieve one-direction electron flow, limiting the potential for back electron flow. Hydrolysis of two ATP molecules associated with the Fe protein occurs after the electron transfer and therefore is not used to directly drive the electron transfer. Rather, ATP hydrolysis is proposed to contribute to relaxation of the "activated" conformational state associated with the ATP form of the complex, with the free energy from ATP hydrolysis being used to pay back energy associated with component protein association and electron transfer. Release of inorganic phosphate (Pi) and protein-protein dissociation follow electron transfer and ATP hydrolysis. The rate-limiting step for the Fe protein cycle is not dissociation of the two proteins, as previously believed, but rather is release of Pi after ATP hydrolysis, which is then followed by rapid protein-protein complex dissociation. Nitrogenase is composed of two catalytic halves that do not function independently but rather exhibit anticooperative nuclear motion in which electron transfer in one-half of the complex partially inhibits electron transfer and ATP hydrolysis in the other half. Calculations indicated the existence of anticooperative interactions across the entire nitrogenase complex, suggesting a mechanism for the control of events on opposite ends of this large complex. The mechanistic necessity for this anticooperative process remains unknown. This Account presents a working model for how all of these processes work together in the nitrogenase "machine" to transduce the energy from ATP binding and hydrolysis to drive N2 reduction.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Diagram of the nitrogenase proteins and the metal-containing cofactors. (left) Schematic representation of the Fe protein component and the MoFe protein component with metal cofactors. (center) Structures of the 4Fe–4S cluster (F), the P-cluster (P), and the FeMo cofactor (M). Structures are from PDB entry 4WZA. (right) Legend showing representations of the metal clusters and oxidation states.
Figure 2.
Figure 2.
Gated electron transfer in nitrogenase. Shown is a schematic of gated electron transfer from the Fe protein to half of the MoFe protein. The electron transfer event is initiated by association of the Fe protein with the MoFe protein (left). In this state, the gate for electron transfer is closed (shown as a blue bar). Next follow protein conformational changes in the complex that result in the uptake of about 80 water molecules (center). The electron transfer gate is opened (shown as two blue lines), allowing electron transfer (red arrow) to occur (right).
Figure 3.
Figure 3.
Updated Fe protein cycle. Shown is the Fe protein with two bound nucleotides and half of the MoFe protein with the P-cluster (P) and the FeMo cofactor (M). The cycle starts at the top left and proceeds clockwise, with the relevant transitions between states and reported rate constants noted on the black arrows. Electron transfers are noted with red arrows. The metal clusters and oxidation states are noted in Figure 1.
Figure 4.
Figure 4.
Scheme illustrating negative cooperativity in the nitrogenase ternary complex. Following protein conformational changes, the electron transfer goes forward in the bottom half (step 1), while the electron transfer does not occur in the top half. Once the bottom half completes electron transfer, ATP hydrolysis, and Pi release, the top half then proceeds through electron transfer and ATP hydrolysis (step 2).

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