Thread by @markherzik

Excited to share our work detailing cryoEM of nitrogenase under catalytic turnover conditions! This collaborative work has enabled the structural characterization of the nitrogenase reaction intermediates at high resolution for the first time. biorxiv.org/content/10.110…

First, I want to say it has been an amazing collaboration with @HannahRutrut (now Ph.D.!), @BCryoEM, @HoangPMNguyen, and @TezcanLab. We paired the best of our strengths to tackle this technically challenging project. Lots of exciting and unexpected results. #teamnitro

Reduced forms of nitrogen are essential for sustaining life. They are required for the biosynthesis of amino and nucleic acids as well as the production of commodity chemicals and fertilizers. Although life can exist without oxygen, life cannot exist without nitrogen.

Nitrogen is present in the environment in various chemical forms including inorganic N2 gas as well as more readily bioavailable organic forms (i.e., NH3). The nitrogen cycle is the process by which nitrogen is converted between chemical forms as it circulates the environment.

The conversion of nitrogen can be carried out through both biological and industrial processes. Importantly, the only enzyme capable of nitrogen fixation is nitrogenase (!!) which catalyzes the 8 electron reduction of atmospheric nitrogen and protons into ammonia and hydrogen.

Nitrogenase is a two-component enzyme consisting of iron protein (FeP, g2 homodimer) and molybdenum-iron protein (MoFeP, a2b2 heterotetramer). Distinct from most redox enzymes, nitrogenase requires ATP hydrolysis for successive transfer of e- and H+ for substrate reduction.

The coupling of ATP hydrolysis to e- transfer is mediated by FeP – the only biological reductant of MoFeP – which forms a nucleotide-dependent complex with MoFep and hydrolyzes two ATPs for each e-/H+ transfer to MoFeP. The part of nitrogenase catalysis is termed the “FeP cycle”.

In the “MoFeP cycle”, the e- from FeP are received by the P-cluster (an [8Fe:7S] complex) of MoFeP and relayed to the iron-molybdenum cofactor (FeMoco, a [7Fe:9S:C:Mo]-homocitrate complex) where N2 reduction occurs. In total, >8 FeP binding events are required for catalysis.

Importantly, ATP-dependent interactions between FeP and MoFeP are necessary for interprotein electron transfer (ET) and for gated ET between the P-cluster and FeMoco.

However, the nature of these long-distance conformational perturbations and directional ET are not understood.

Despite previous crystallographic studies, these static views – obtained under non-turnover conditions – showed no variation in the structure of MoFeP, providing little insight into the mechanism of ATP/FeP-mediated redox events within nitrogenase.

Nitrogenase meet #cryoEM ;)

So how does ATP hydrolysis drive N2 fixation? Using cryoEM we visualized for the first time nitrogenase under catalytic turnover. Our structures show that asymmetry governs all aspects of nitrogenase mechanism – ATP hydrolysis, protein-protein interactions, and catalysis.

As a reference, we obtained a ~1.8-Å resolution cryoEM structure (1.63 Å Nyquist) of resting-state Azotobacter vinelandii MoFeP, termed “rsMoFeP”, where we could unambiguously determine the location of atoms in the metal clusters as well as the oxidation state.

We then collected 15K cryoEM movies (20M particles) of nitrogenase under high-electron flux turnover conditions (10-fold molar excess of FeP over MoFeP and high MgATP) to maximize NH3 production. All samples were prepared anaerobically and manually frozen jove.com/v/62765/manual…

Unexpectedly, contrary to previous X-ray studies, we did not observe any 2:1 FeP:MoFeP complexes – only 1:1 complexes or free MoFeP or FeP. Given the C2 symmetry of MoFeP, it had long been assumed that each MoFeP “half” operates independently, with each able to bind a FeP.

To ensure our cryoEM sample prep did not bias 1:1 complexes, we prepared a second set of turnover sample with BeFx which is known to arrest nitrogenase in quasi-irreversible 2:1 and 1:1 FeP:MoFeP complexes. Accordingly, our cryoEM data contained largely 2:1 FeP:MoFeP complexes.

So what is the difference in the two turnover 1:1 FeP:MoFeP complexes? Turns out we were able to isolate complexes where FeP was bound to ATP/ATP or after a single ATP hydrolysis event yielding ATP/ADP. Not only is the FeP:MoFeP complex asymmetric, but ATP hydrolysis is as well!

Conformational changes in FeP during ATP hydrolysis and a short [4Fe:4S]-to-P-cluster distance of ~15 Å prime nitrogenase for rapid interprotein ET. We expect these asymmetric conformational changes are critical for timing interprotein ET when FeP is bound in the activated state.

Principal component analysis of FeP from the turnover complexes compared to twenty available free- or MoFeP-complexed FeP structures (X-ray) detail concerted hinging (86%) and twisting (6%) motions within FeP concurrent with nucleotide state and hydrolysis.

Are there conformational changes in MoFeP? Shockingly not at the half of MoFeP bound to FeP (proximal). But there are intriguing rotameric changes surrounding the distal MoFeP metal sites that render MoFeP asymmetric. And these are right next to FeMoco – where catalysis occurs.

Although these changes are subtle, they result in alterations in the proposed substrate pathway, termed the IS channel, that lead from the surface of MoFeP to FeMoco (where catalysis occurs). During turnover, we observe this pathway is diverted to different faces of FeMoco.

So what’s happening at FeMoco? Interestingly, not much at the proximal FeMoco when compared to rsMoFeP. However, at the distal FeMoco site there are changes in the FeMoco local environment as well as FeMoco itself that indicate it is more dynamic than previously anticipated.

Our observations provide evidence that asymmetry governs all aspects of nitrogenase function, from ATP hydrolysis to protein-protein interactions and e- transfer. Given the complexity of nitrogenase, it is expected numerous subtle concerted changes coordinate nitrogen reduction.