Probing the All-Ferrous States of Methanogen Nitrogenase Iron Proteins

Abstract

The Fe protein of nitrogenase reduces two C1 substrates, CO 2 and CO, under ambient conditions when its [Fe 4 S 4 ] cluster adopts the all-ferrous [Fe 4 S 4 ] 0 state. Here, we show disparate reactivities of the nifH - and vnf -encoded Fe proteins from Methanosarcina acetivorans (designated Ma NifH and Ma VnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO 2 and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that Ma VnfH adopts the all-ferrous state at a more positive reduction potential than Ma NifH, which could account for the weaker reactivity of the Ma VnfH toward C1 substrates than Ma NifH. More importantly, Ma VnfH already displays the g = 16.4 parallel-mode EPR signal that is characteristic of the all-ferrous [Fe 4 S 4 ] 0 cluster at a reduction potential of −0.44 V, and the signal reaches 50% maximum intensity at a reduction potential of −0.59 V, suggesting the possibility of this Fe protein to access the all-ferrous [Fe 4 S 4 ] 0 state under physiological conditions. These results bear significant relevance to the long-lasting debate of whether the Fe protein can utilize the [Fe 4 S 4 ] 0/2+ redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes.

A complete biomimetic iron-sulfur cubane redox series

“…Recent studies suggested that the ability of the FeP to access the superreduced state in operando enables the enzyme to operate at twice the efficiency in terms of ATP consumed per electron transferred than when operating with the [Fe4S4] 1+/2+ couple (2,14). In addition, reduction to the all-ferrous state of the FeP isolated from Methanosarcina acetivorans was shown to catalyze the conversion of C1 substrates such as CO2 and CO into hydrocarbons under ambient conditions (15). Last, as a biological synthon, the superreduced FeS cubane has been proposed to play a role during the maturation of the nitrogenase P-cluster (16,17).…”

Nitrogenase Fe Protein: A Multi-Tasking Player in Substrate Reduction and Metallocluster Assembly

“…This observation implies that the Fe protein could potentially access the all-ferrous state under certain physiological conditions. Consistent with this suggestion, it was reported that the vnfH -encoded Fe protein (VnfH) from Methanosarcina acetivorans displayed 50% of maximum intensity of the all-ferrous specific EPR signal at −0.59 V, a potential well within the range of the physiological reduction potentials within the cell [ 19 ]. The “pink all-ferrous state” of the Fe protein was further correlated with the ability of this protein to catalyze the reduction of C 1 substrates (see Section 3.3 below); however, experimental evidence is yet to be acquired to address whether this all-ferrous state could be used to drive nitrogenase catalysis under in vivo conditions.…”

“…However, studies in the last decade have revealed that other than functioning as the obligate electron donor for the MoFe protein during nitrogenase catalysis ( Figure 2 a), the Fe protein of the Mo-nitrogenase also plays a key role in the assembly of both complex metalloclusters of its catalytic partner, serving as a Mo/homocitrate insertase for the maturation of the M-cluster ( Figure 2 b) [ 12 , 13 , 14 ] as well as an ATP-dependent reductase for the maturation of the P-cluster ( Figure 2 c) [ 14 , 15 , 16 , 17 , 18 ]. Moreover, the homologous Fe proteins of both bacterial and archaeal origins were shown to act as reductases on their own and catalyze the reduction of CO 2 and/or CO to hydrocarbons ( Figure 2 d) [ 19 , 20 , 21 , 22 , 23 , 24 ] These findings not only establish the Fe protein as a crucial multi-tasking player in nitrogenase catalysis and assembly but also point to the utility of this protein as a simple FeS template for the future development of biotechnological applications that recycle CO 2 (a greenhouse gas) and CO (a carbon waste) into the useful hydrocarbon products.…”

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4Se4] Cluster

“…To circumvent this problem, we treated AvNifH and AvNifH Se with 20 mM dithionite (E 1/2 = À 0.44 V at pH 8.0), 2 mM dithionite (E 1/2 = À 0.47 V at pH 8.0), 10 mM europium(II) 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10tetraazacyclododecane (Eu II -DOTAM; E 1/2 = À 0.59 V at pH 8.0), 10 mM europium(II) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Eu II -DOTA; E 1/2 = À 0.92 V at pH 8.0), and 10 mM europium(II) diethylenetriamine pentaacetate (Eu II -DTPA; E 1/2 = À 1.14 V at pH 8.0), and monitored the intensity of the all-ferrous, g = 16.4 parallel-mode EPR signal at the various reduction potentials created by these reductants (Figure 6b). [16,37] In the case of AvNifH, the g = 16.4 signal are absent from the EPR spectra at À 0.44 V (i.e., 20 mM dithionite) and À 0.47 V (i.e., 2 mM dithionite); whereas in the case of AvNifH Se , the intensities of the g = 16.4 signal are 4 % and 13 %, respectively, of the maximum intensity at À 0.44 V and À 0.47 V. Plotting of the intensity of the g = 16.4 signal versus the reductant potentials allowed for an approximate assignment of the "mid-intensity" potential, or the potential corresponding to 50 % of the maximum signal intensity, for AvNifH and AvNifH Se (Figure 6c). Strikingly, the "mid-intensity" potential for AvNifH is � À 0.90 V, which approximates the midpoint potential of À 0.79 V previously determined for the [Fe 4 S 4 ] 1 + /0 couple by a redox mediator-based method.…”