[1S03m] 06. Enzyme Engineering/Enzymology - Part 1

11:15 AM - 11:30 AM JST(2:15 AM - 2:30 AM UTC)

[1ENZ-04] Structural basis of redox-tuning in an iron-sulfur protein

○Faisal Hammad Mekky Koua¹, Tiankun Zhou², Tokushi Sato¹, Jayanath Koliyadu^1,3, Marcin Sikorski¹, Henry Chapman^3,4, Richard Bean¹, Adrian Mancuso^1,2 (1. European X-Ray Free Electron Laser Facility (Germany), 2. Diamond Light Source (DLS) (UK), 3. Center for Free Electron Laser Science (CFEL), DESY (Germany), 4. University of Hamburg (Germany))

Keywords:

Metalloenzymes,Redox-tuning,Iron-sulfur proteins,Electron transfer,Sub-Ångström resolution serial femtosecond crystallography

Understanding fine structural dynamics of metalloenzymes at near-physiological temperatures, with high spatial and temporal resolution, is essential to elucidating the structural basis of redox tuning (1–3). Here, using serial femtosecond crystallography with ultrashort X-ray pulses (~25 fs and ~15 keV) at room temperature (rt-SFX), we resolved a chemically-reduced state structure of a high-potential iron sulfur protein (HiPIP) from the photosynthetic bacterium Thermochromatium tepidum at an ultra-high resolution (0.98–1.05 Å), the first room-temperature sub-Å resolution structure at XFEL sources. This enabled us to obtain a damage-free and accurate model of the 4Fe–4S cluster—a ([4Fe–4S](S_γ-Cys)₄) cluster—and resolve fine chemical structures of the nearby hydrogen-bonding network crucial for redox-tuning. Additionally, we resolved high-resolution structures (~1.5 Å) of the oxidized state under both cryogenic and room-temperature conditions. Our results reveal key differences between these redox states particularly at the cluster site. Intriguingly, most of the intra-atomic distances of the 4Fe–4S cluster (reduced state) are shortened by 0.03–0.1 Å in comparison with the cryogenic synchrotron structure, indicating that the rt-SFX model is less affected by radiation-damage. Furthermore, by comparing our reduced structure with the active (oxidized) state (3, 4), we observed critical differences in the amide protons of key residues within the cluster vicinity, suggesting a vital role in redox-tuning of HiPIP in bacterial photosynthesis. Consistent with this, our isomorphous difference map reveals highly localized density features at the 4Fe–4S cluster and its immediate environment. Altogether, our results provide a blueprint for redox-tuning during metalloenzyme catalysis, paving the way for engineering functionally optimized biocatalysts.

References
[1] Hirano, Y., Takeda, K. & Miki, K. Nature 534, 281–284 (2016).
[2] Dey, A., et al. Science 318, 1464–1468 (2007).
[3] Hanazono, Y., et al. Sci. Adv. 8, eabn2276 (2022).
[4] Ohno, H, et al. PLoS ONE 12, e0178183 (2017).