Tuning Electron Flux through Nitrogenase with Methanogen Iron Protein Homologues

Abstract

Nitrogenase uses a reductase component called Fe protein to deliver electrons to its catalytic partner for substrate reduction. The essential role of Fe protein in catalysis makes it an ideal target for regulating the electron flux and enzymatic activity of nitrogenase without perturbing the cofactor site. This work reports that hybrids between the Fe protein homologs of Methanosarcina acetivorans and the catalytic components of Azotobacter vinelandii can trap substrate CO through reduced electron fluxes. In addition, homology modeling/in silico docking is used to define markers for binding energy and specificity between the component proteins that correlate with the experimentally determined activities. This homologue-based approach could be further developed to allow identification or design of hybrids between homologous nitrogenase components for mechanistic investigations of nitrogenase through capture of substrates/ intermediates or for transgenic expression of nitrogenase through synthetic biology.

The Fe Protein: An Unsung Hero of Nitrogenase

“…Detailed analysis of a synthetic all-ferrous [Fe 4 S 4 ] cluster by Mössbauer, EPR and density functional theory (DFT) calculations, in comparison to the data obtained for the [Fe 4 S 4 ] 0 NifH species, helped to establish that the ground state configuration for NifH is S = 4 [39][40][41]. Moreover, the diagnostic color and signal has been observed in both super-reduced bacterial and archaeal Fe protein variants [42], indicating that access of this state is conserved among Fe proteins. Identification of a doubly reduced Fe protein suggests that perhaps the system could transfer two electrons per two ATP as a means to render a more efficient electron transfer during catalysis.…”

Strategies Towards Capturing Nitrogenase Substrates and Intermediates via Controlled Alteration of Electron Fluxes

“…Further research along this line will involve screening of other low‐potential electron donors and Fe protein homologs for candidates that can be used to trap other important substrates, such as N 2 , as well as the reaction intermediates of N 2 and CO reduction. With regard to strategy 2, the screening of Fe protein homologs could be facilitated by a homology modeling/docking approach we developed recently and tested on four Fe protein candidates . While further verification is required for establishing the validity of this approach, the outcome of the initial test was promising, and automation of this screening method could be attempted in combination with protein design calculations and in silico mutant screening to further modulate the electron donating capacity of the Fe protein.…”

“…It can be reasoned that a mismatch between a homologous, yet distinct Fe protein from one organism and the catalytic component from another could result in a reduced electron flux through the hybrid system, thereby ‘stalling“ the reaction and facilitating the capture of substrate or reaction intermediates at the cofactor site. Consistent with this argument, when the reductase component, VnfH, from Methanosarcina acetivorans (designated VnfH Ma ) was paired with the catalytic component, VnfDGK, from A. vinelandii (designated VnfDGK Av ), there was a significant decrease of substrate reduction activity that reflected a dramatic decrease in the electron flux through this hybrid system; in particular, the activity of CO reduction was nearly abolished, suggesting a possible trapping of CO in an unactivated state at the cofactor site (Figure a) …”

“…Indeed, EPR analysis revealed accumulation of CO on VnfDGK Av through limited turnover by VnfH Ma /VnfDGK Av hybrid, which resulted in a complex EPR signal (designated hi‐CO B ) practically indistinguishable from that of the hi‐CO A state generated upon incubation of VnfDGK Av with Eu II ‐DTPA and 2.6 atm CO (Figure b) . This observation was exciting, as it established the relevance of the hi‐CO conformation of V‐nitrogenase to turnover conditions and suggested the possible presence of two types of CO‐binding sites on the cofactor: one with high affinity for catalytically competent binding of CO (lo‐CO); and one with low affinity for catalytically incompetent binding of CO (extra‐CO).…”

“…(a) Structure and function of V‐nitrogenase. Shown are the energy‐optimized homology model of the reductase component (upper, encoded by vnfH ) and the crystal structure of one half of the catalytic component (lower, encoded by vnfDGK ) of the V‐nitrogenase of A. vinelandii . During catalysis, the reductase (VnfH Av ) and catalytic component (VnfDGK Av ) for a functional complex, which allows electrons to flow from the [Fe 4 S 4 ] cluster of former, via the P*‐cluster to the V‐cluster of the latter, where substrate reduction occurs.…”