H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle

Abstract

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron‑sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.

The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+

“…S13). Moreover, in Nfn, the subsequent oxidation of ASQ-BF-FAD by reduction of ET-FAD is gated by an intervening [2Fe-2S] cluster, which mediates electron flow via the ET-FAD to the high-potential acceptor (in this case, NAD ϩ ) in the absence of any significant conformational change (56). In further contrast, electron flow to the high-potential pathway in EtfABCX is gated by a flavin 1 e Ϫ acceptor and a rather dramatic conformational change that enables oxidation of ET-FAD (31).…”

Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins

“…For every time point, 10 μl must be available to add to the quench solution, plus an additional 10 μl for the 24 h/fully deuterated time point. For instance, in the HDX experiment of Nfn, five time points were measured (1 min, 3 min, 15 min, 60 min, 3 h, and 24 h) (Berry et al ., 2018). An additional 10 μl of reaction solution is included to prevent withdrawing the full reaction solution, thus bringing the total volume to 70 μl.…”

“…FBEB generates reduced ferredoxin, which can be oxidized to produce energy. Proteins that are capable of reducing ferredoxin are of great interest and have been the focus of recent studies using HDX-MS (Demmer et al ., 2016; Lubner et al ., 2017; Berry et al ., 2018). HDX-MS is a powerful technique for investigating protein stability, dynamics, and ligand binding providing information about the relationship between structure and function.…”

“…The Mass Spectrometry Facility at MSU was supported in part by the Murdock Charitable Trust and an NIH IDEA program grant P20GM103474. The work detailed in this protocol was adapted from work described in recent publications (Lubner et al ., 2017; Berry et al ., 2018). The authors declare that they do not have any conflicts of interest.…”

Residue-Level Allostery Propagates Through the Effective Coarse-Grained Hessian

“…There are many well established experimental techniques for characterizing allostery, including activity based assays to investigate non-Michaelis-Menten kinetic behavior, 19 H/D mass spectrometry, 20,21 as well as structural based approaches such as X-ray crystallography, 22 cryoEM, 23 and NMR. [24][25][26][27][28][29][30][31][32] Structural techniques can be used to identify residues that interact directly with or are structurally perturbed by the e↵ector molecule by comparing apo and e↵ector bound states of a protein.…”