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Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur 'P' clusters.

ABSTRACT: Hydrolysis of MgATP to MgADP is essential for nitrogenase action. There is good evidence for binding of both nucleotides to the Fe protein of nitrogenase, but data indicating their binding to the MoFe protein have been controversial [see Miller and Eady (1989) Biochem. J. 263, 725-729]. The binding of MgADP to the MoFe protein of nitrogenase of Klebsiella pneumoniae was investigated by non-equilibrium gel-filtration column methods. No binding of MgADP to the dithionite-reduced protein could be detected. Treatment of the MoFe protein with phenosafranine [midpoint potential (Em) -270 mV] did not affect the activity, and oxidized the 'P' clusters but not the iron-molybdenum cofactor (FeMoco) centres. This oxidized species bound 3.9 mol of MgADP with a binding pattern characteristic of low rates of ligand dissociation. These observations suggest that the variability in published data on nucleotide binding to the MoFe protein is related to poor control of the protein oxidation level. Our data, coupled with the observation that 'P' clusters become oxidized during reduction of N2 [Lowe, Fisher and Thorneley (1993) Biochem. J., in the press], led us to propose that the ADP binding sites are transiently filled during enzyme turnover by hydrolysis of ATP originally bound to the Fe protein, and that hydrolysis occurs on a bridging site on the MoFe-Fe-protein complex.

SUBMITTER: Miller RW

PROVIDER: S-EPMC1132426 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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Project description: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.

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Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur 'P' clusters.

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