Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

Abstract

The biological reduction of N 2 to NH 3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured firstorder rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: The ATP-driven reduction of one N 2 with evolution of one H 2 requires a minimum of 8 e − and the hydrolysis of 16 ATP molecules in a complex cascade of events in which electron transfer (ET) from the nitrogenase Fe protein to the catalytic MoFe protein is coupled to the hydrolysis of two ATP molecules (1, 3, 4). The Fe protein is a homodimer with a single [4Fe-4S] cluster and two nucleotide binding sites, one in each subunit (5). The MoFe protein is an α 2 β 2 -tetramer, with each αβ-pair functioning as a catalytic unit that binds an Fe protein (6). Each αβ-unit contains an [8Fe-7S] cluster (abbreviated as P cluster) and a [7Fe-9S-Mo-C-R-homocitrate] cluster (abbreviated as FeMo cofactor or M cluster) (6-10). In each ET event, the Fe protein, in the reduced (1+) state with two bound ATP, first associates with the MoFe protein (Fig. 1). In a recent model, termed "deficit spending," it is proposed that this association triggers a two-step ET event (11, 12). The first ET step occurs inside the MoFe protein, involving ET from the P cluster resting state (P N ) to the resting FeMo cofactor (M N ), resulting in an oxidized P cluster (P 1+ ) and a reduced FeMo cofactor (M R ) (12). This ET event is conformationally gated (11) with an apparent first-order rate constant (k ET ) between 100 and 140 s −1 (11, 12). In the second ET step, an electron is transferred from the Fe protein [4Fe-4S] cluster to the oxidized P 1+ cluster, resulting in the return of the P cluster to the resting oxidation state (P N ) and an oxidized 2+ cluster in the Fe protein (12). This second step is fast, having a rate constant greater than 1,700 s −1 (12). Transfer of one electron from the Fe protein to an αβ-unit of MoFe protein is known to be coupled to the hydrolysis of the two ATP molecules bound to the Fe protein, yielding two ADP and two Pi (2). Following the hydrolysis reaction, the two phosphates (Pi) are released from the protein complex with a first-order rate constant (k Pi ) of 22 s −1 at 23°C (13 Although the energetic coupling between ET and ATP hydrolysis is firmly established (1, 3, 4, 16), the nature of this coupling has remained unresolved: does ATP hydrolysis itself provide the principal energy input for the conformational change (s) that drive ET from Fe protein to the MoFe protein, or, does the bound ATP induce the formation of a reactive, "activated" conformation of the complex, with E...

Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

“…3). An exponential rise to maximum is observed, with a rate constant of k ET ∼140 s −1 , in agreement with earlier reports (12,13), and our finding that ET precedes ATP hydrolysis (discussed above) and P i release in the "Fe-protein" cycle (11). At later times, in steady state, this absorbance difference actually decreases as released Fe ox is rereduced and the system passes into a steady-state ratio of Fe ox /Fe red .…”

“…The presteady-state rate constant is roughly half the value we reported earlier (11), with the difference arising from a more efficient quenching of the reaction by formic acid, rather than the EDTA quench used previously. The new value nonetheless remains consistent with the key finding of the earlier study: ATP hydrolysis follows ET (11). The rate constant for the linear phase (12 s −1 ) is consistent with the turnover number of nitrogenase for substrate reduction with dithionite as the reductant (21).…”

“…P i Release. P i release was reported to occur with a rate constant of ∼17 s −1 , consistent with this release following ET and ATP hydrolysis (11). This process has now been reexamined quantitatively.…”

“…The end result of this cycle is the transfer of an electron from the Fe protein to the FeMo-co and hydrolysis of the two ATP molecules. The order of the key steps during this cycle has only been established recently (11). The first step is electron transfer (ET) with an observed rate constant of k ET ∼140 s −1 (12,13).…”

“…This process has been shown to occur in two steps: a conformationally gated ET from the P cluster to FeMo-co followed by rapid reduction of the oxidized P cluster by the Fe red protein, with the two steps collectively referred to as "deficit spending" ET (14). ATP hydrolysis then follows with an observed rate constant of k ATP ∼70 s −1 , followed by events associated with P i release, which occur with an overall rate constant k Pi ∼16-25 s −1 (11,15,16). This is followed by dissociation of the oxidized Fe ox (ADP) 2 protein from the MoFe protein, which is seen to occur with a rate constant k diss >700 s −1 when flavodoxin is the reductant, to complete the Fe protein cycle (16).…”