Reduction of nitrogenase Fe protein from Azotobacter vinelandii by dithionite: quantitative and qualitative effects of nucleotides, temperature, pH and reaction buffer

Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle

“…Additionally, these studies were conducted at 4 °C to slow the reactions enough to observe them. Consistent with previous studies, nucleotides significantly slow the rates of reduction of Fe ox protein by DT (by ∼100-fold). , In contrast, with Fld HQ as the reductant, the rates of reduction remained fast and roughly unchanged with or without nucleotide present (Figure ). , …”

“…Consistent with previous studies, nucleotides significantly slow down the rates of reduction of Fe ox protein by DT (by ~ 100-fold). 22,23 In contrast, with Fld HQ as reductant, the rates of reduction remained fast and roughly unchanged with or without nucleotide present ( Figure 5). 25,27 Taken together, the studies presented so far suggest that when Fld HQ is the reductant, the Page 19 of 44 ACS Paragon Plus Environment Biochemistry 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 4 0 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5 5 6 5 7 5 8 5 9 6 0 20 Fe ox (ADP)2 dissociation from the MoFe protein is not rate limiting and does not correspond to the overall kcat for substrate reduction.…”

“…The studies, to this point, favor rapid dissociation of the Fe ox (ADP) 2 protein from the MoFe protein when Fld HQ is the reductant. The observed lower rate of dissociation when DT is the reductant could indicate slow reduction of Fe ox protein, rather than slow dissociation of the Fe protein from the MoFe protein. ,, Several previous studies have illustrated low activity for nitrogenase at low a concentration of SO 2 •– as the reductant. − ,, To determine the rates of reduction of Fe ox protein by different reductants, the pre-steady-state studies were conducted using Fe ox in the absence and presence of a nucleotide (ADP or ATP). Fe ox protein in one syringe was rapidly mixed against either DT or Fld HQ in the other syringe.…”

“…Over the past five decades, most of the in vitro mechanistic studies of nitrogenase have been conducted with the nonphysiological reductant dithionite (DT), largely because of ease of use. ,, The kinetic parameters for each step in the Fe protein cycle using DT as a reductant have been deduced and are summarized in the scheme shown in Figure . − ,, It was concluded from a comparison of the rate constants that the overall rate-limiting step in the Fe protein cycle is the dissociation of Fe ox (ADP) 2 from the MoFe protein with a rate constant of ∼6 s –1 . , A problem that is often overlooked in such studies is the reversible dissociation of DT (S 2 O 4 2– ) to generate the actual reductant, the radical anion SO 2 •– , with a K d of ∼1.5 nM and a slow rate constant of ∼2 s –1 . This leads to slow reduction of Fe ox , with rate constants for this reduction near the rate constant that is reported for dissociation of Fe ox (ADP) 2 from MoFe protein. ,,, …”

Flavodoxin hydroquinone reduces Azotobacter vinelandii Fe protein to the all-ferrous redox state with a S = 0 spin state

“…1 0 Av2(MgAXP), then that would yield an ATP͞2e -ratio of 2.54, measurably higher than the observed lower limit of 2.0, suggesting that the remaining Av2(MgAXP) 2 may be reduced to the [Fe 4 S 4 ] 0 state in slower phases of reduction. In particular, nucleotides are known to lower the rate of reduction of Av2 by DT (37)(38)(39)(40)(41)(42), even breaking up the reduction into multiple phases, some fast, some slow.…”