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...
Rdc2 is the first flavin-dependent halogenase identified from fungi. Based on the reported structure of the bacterial halogenase CmlS, we have built a homology model for Rdc2. The model suggests an open substrate binding site that is capable of binding the natural substrate, monocillin II, and possibly other molecules such as 4-hydroxyisoquinoline (1) and 6-hydroxyisoquinoline (2). In vitro and in vivo halogenation experiments confirmed that 1 and 2 can be halogenated at the position ortho to the hydroxyl group, leading to the synthesis of the chlorinated isoquinolines 1a and 2a, respectively, which further expands the spectrum of identified substrates of Rdc2. This work revealed that Rdc2 is a useful biocatalyst for the synthesis of various halogenated compounds.
The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3' ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.
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