Cryo-EM structures of photosystem I with alternative quinones reveals new insight into cofactor selectivity

This study presents high-resolution cryo-EM structures of Photosystem I from a *Synechocystis* Δ*menB* variant containing alternative quinones, revealing unexpected asymmetry in quinone binding and exchange between the A1A and A1B sites and providing new insights into cofactor selectivity and metabolic plasticity.

The Gist

Imagine a solar-powered factory called Photosystem I. Its job is to take sunlight and turn it into chemical energy, kind of like a battery charger for a living cell. Inside this factory, there are two specific "charging ports" (called the A1A and A1B sites) where special molecules called quinones plug in to help move electricity.

Normally, these ports are designed to accept a very specific type of plug: a long, sturdy, custom-made plug called Phylloquinone.

The Experiment: Breaking the Factory

The scientists in this study decided to play a game of "what if." They genetically modified a tiny algae (cyanobacteria) so it couldn't make its own custom plugs anymore. This is like taking a factory and cutting off the supply line for its special batteries.

Without the custom plugs, the factory had to improvise. It started using whatever spare parts were lying around in the cell, specifically a different kind of plug called Plastoquinone-9 (PQ-9). This plug is similar but has a very long, floppy tail (like a long, wiggly extension cord) compared to the short, neat tail of the custom plug.

The Big Discovery: One Door is Stuck, One is Open

For years, scientists thought these two charging ports (A1A and A1B) were identical twins—they would both happily swap out their plugs for the new, wiggly PQ-9.

But when the scientists built a super-high-resolution 3D map of this factory using a powerful microscope (Cryo-EM), they found something surprising: The two ports are not twins at all.

• The A1A Port (The "Open Door"): This port is on the edge of the factory. It's loose and flexible. It happily accepted the long, wiggly PQ-9 plug. Because the plug is so floppy, it makes the surrounding machinery a bit messy and hard to see in the microscope. But the best part? You can easily pull this PQ-9 out and swap it for a brand new, custom-made plug (like the one they added in the experiment, called ENQ).
• The A1B Port (The "Stuck Door"): This port is deep in the center of the factory, right where the different parts of the machine are glued together. It turns out this port is picky. It mostly rejected the long, wiggly PQ-9 and stuck with a shorter, neater plug (DMPBQ) that the factory accidentally made on its own. Because this plug fits perfectly and is short, the machinery around it is very stable and clear. Crucially, you cannot easily swap this plug out. It's stuck.

The "Tail" Analogy

Think of the quinone plugs like umbrellas.

• The custom plug is a compact, short-handled umbrella.
• The PQ-9 plug is a giant beach umbrella with a 45-foot pole.

The scientists found that the A1A port is like an open-air patio. You can shove that giant beach umbrella in there, and even though it wiggles around and blocks the view, you can still pull it out and swap it for a different umbrella.

The A1B port is like a tight, narrow hallway in the middle of a crowded building. If you try to shove that giant beach umbrella in there, it gets stuck and jams the whole hallway. So, the factory naturally prefers a short, compact umbrella there. It's so tight that you can't swap it out without tearing the building apart.

Why Does This Matter?

This study solves a mystery that has been around for 20 years. Scientists knew they could swap plugs in these algae, but they didn't know how or why it worked.

1. It explains the "messiness": The reason the A1A port looked blurry in the microscope was because the long, wiggly tail of the PQ-9 plug was flopping around, making the surrounding proteins shake.
2. It reveals a design flaw (or feature): The factory is actually a bit unstable when it uses the wrong plug in the wrong spot. The long tail in the A1A port makes that side of the machine wobbly.
3. It helps future engineering: If we want to build better solar cells or bio-fuels using these natural machines, we need to know which parts are flexible and which are rigid. We now know that if we want to swap parts, we have to target the "open door" (A1A), not the "stuck door" (A1B).

The Bottom Line

Nature is clever, but it's also messy. When you break a part of a biological machine, it doesn't just stop; it improvises. This paper shows us that even though the two charging ports look the same from the outside, they behave very differently inside. One is a flexible, swap-out zone, and the other is a rigid, locked-down zone. Understanding this difference helps us learn how to fix and improve the solar engines of life.

Technical Summary

1. Problem Statement

Photosystem I (PS I) is a critical photooxidoreductase in oxygenic photosynthesis, utilizing high-affinity quinones (typically phylloquinone, PhQ) in its $A_1$ binding sites ($A_{1A}$ and $A_{1B}$) for electron transfer. To study quinone chemistry and engineer PS I for bioenergy applications, researchers utilize the cyanobacterium Synechocystis sp. PCC 6803 with a disrupted menB gene ($\Delta menB$). This mutation blocks native PhQ biosynthesis, forcing the incorporation of exchangeable plastoquinone-9 (PQ-9) or exogenous naphthoquinones.

Despite decades of biophysical studies on the $\Delta menB$ variant, a critical gap remained: the lack of high-resolution structural data explaining how these alternative quinones bind, their occupancy levels, and why the $A_{1A}$ and $A_{1B}$ sites behave differently regarding ligand exchange. Previous structural work on a "laboratory-evolved" $\Delta menB$ strain ($\Delta menB(2023)$) showed stable, non-exchangeable quinones, failing to provide insight into the dynamic exchange observed in the original strains.

2. Methodology

The authors employed a multi-disciplinary approach combining genetics, biochemistry, cryo-electron microscopy (cryo-EM), and spectroscopy:

• Strain Generation: They utilized a newly generated $\Delta menB(2024)$ strain, which recapitulates the original $\Delta menB(2002)$ phenotype. This strain grows under low light and retains the ability to exchange quinones, unlike the evolved 2023 strain.
• Sample Preparation:
  • PS I$\Delta menB$: Purified from the $\Delta menB(2024)$ strain containing endogenous PQ-9 and DMPBQ.
  • PS I$\Delta menB$+ENQ: The purified complex was incubated with exogenous 2-ethyl-1,4-naphthoquinone (ENQ) to displace native quinones.
• Cryo-EM Structure Determination:
  • Single-particle cryo-EM was used to determine structures at 1.90 Å (PS I$\Delta menB$) and 2.05 Å (PS I$\Delta menB$+ENQ) resolution.
  • Map Analysis: The authors analyzed electron density maps for quinone headgroups and tails. They utilized Q-scores to quantify local structural order and flexibility.
  • Occupancy Estimation: A custom computational procedure was developed to deconvolute mixed quinone populations by sampling map intensity along specific carbon atoms of the quinone headgroups.
• Spectroscopic Validation:
  • LC/MS: Verified the specific quinone species (PhQ, PQ-9, DMPBQ, ENQ) present in the samples.
  • Time-Resolved EPR (TR-EPR): Performed on the samples to characterize the radical pair states ($P_{700}^+ A_1^-$) and confirm quinone binding orientations, comparing the new strain data with historical data from the 2002 strain.
• Native Gel Electrophoresis: Used to assess the monomer-to-trimer ratio of PS I complexes to correlate structural stability with quinone occupancy.

3. Key Results

A. Asymmetric Quinone Occupancy and Exchangeability

Contrary to the long-held assumption that $A_{1A}$ and $A_{1B}$ sites are symmetric and equally exchangeable, the study revealed significant asymmetry:

• $A_{1A}$ Site (A-side): Primarily occupied by PQ-9 (C-45 tail) in the native state. This site is highly flexible (low Q-scores) and fully exchangeable. Upon ENQ incubation, PQ-9 is displaced by ENQ. The tail density is disordered, indicating the long C-45 tail creates local instability.
• $A_{1B}$ Site (B-side): Primarily occupied by DMPBQ (C-19 tail, similar to native PhQ) in the native state. This site is structurally ordered (high Q-scores) and largely non-exchangeable. However, a minority fraction (~35%) of $A_{1B}$ sites contains PQ-9 or is empty, allowing for partial exchange with ENQ.

B. Structural Basis for Exchangeability

• Tail Length and Disorder: The long, flexible C-45 tail of PQ-9 in the $A_{1A}$ site causes disorder in surrounding subunits (PsaF, PsaJ, and specific chlorophylls), lowering local resolution and facilitating exchange.
• Stability via Tail Length: The shorter C-19 tail of DMPBQ in the $A_{1B}$ site fits tightly within a constrained environment near the trimer interface, stabilizing the complex and preventing exchange.
• Trimerization: Blue native gel analysis showed that the $\Delta menB(2024)$ strain has a higher monomer-to-trimer ratio than Wild Type. The authors hypothesize that the presence of long-tail PQ-9 in the $A_{1B}$ site (which is near the monomer-monomer interface) destabilizes trimer formation, whereas the $A_{1A}$ site (peripheral) tolerates PQ-9 without disrupting trimerization.

C. Quinone Orientation and Binding

• ENQ Binding: In the $A_{1A}$ site, ENQ binds primarily in a "flipped" orientation (75% population) relative to the native PhQ tail, with a minor "unflipped" population (25%). This structural finding confirms previous TR-EPR interpretations regarding hyperfine coupling patterns.
• Mixed Populations in $A_{1B}$: In the $A_{1B}$ site of the ENQ-incubated sample, the occupancy is mixed: ~66% DMPBQ/PQ-9, ~25% unflipped ENQ, and ~10% flipped ENQ.

4. Significance and Impact

• Resolution of a Decades-Old Mystery: The study provides the first high-resolution structural explanation for why the $\Delta menB$ variant exhibits quinone exchangeability. It proves that exchangeability is not uniform across the two binding sites but is dictated by local structural constraints and quinone tail length.
• Mechanism of Ligand Selectivity: It establishes that protein instability is a prerequisite for ligand exchange. The $A_{1A}$ site allows exchange because the long PQ-9 tail induces disorder; the $A_{1B}$ site resists exchange because the shorter DMPBQ tail stabilizes the protein matrix.
• Implications for Bioengineering: Understanding the structural basis of quinone selectivity is crucial for engineering PS I for biohybrid systems (e.g., hydrogen production). It suggests that to incorporate specific exogenous quinones, one must account for the steric and stability constraints of the specific binding pocket ($A_{1A}$ vs. $A_{1B}$).
• Evolutionary Insight: The findings parallel observations in Photosystem II (PS II), where the stable $Q_A$ site (near the interface) holds a tightly bound quinone, while the peripheral $Q_B$ site holds a mobile, exchangeable quinone. This suggests a conserved evolutionary strategy in reaction centers regarding cofactor stability and exchange.

In summary, this work bridges the gap between biophysical data and structural biology, revealing that the "plasticity" of the $\Delta menB$ variant is a result of asymmetric structural stability driven by quinone tail length, fundamentally altering the understanding of cofactor selectivity in Photosystem I.