X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding

Darren J. Hsu, Denis Leshchev, Dolev Rimmerman, Jiyun Hong, Matthew S. Kelley, Irina Kosheleva, Xiaoyi Zhang, Lin X. Chen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


Cytochrome c (cyt c) has long been utilized as a model system to study metalloprotein folding dynamics and the interplay between active site ligation and tertiary structure. However, recent reports regarding the weakness of the native Fe(ii)-S bond (Fe-Met80) call into question the role of the active site ligation in the protein folding process. In order to investigate the interplay between protein conformation and active site structures, we directly tracked the evolution of both during a photolysis-induced folding reaction using X-ray transient absorption spectroscopy and time-resolved X-ray solution scattering techniques. We observe an intermediate Fe-Met80 species appearing on ∼2 μs timescale, which should not be sustained without stabilization from the folded protein structure. We also observe the appearance of a new active site intermediate: a weakly interacting Fe-H2O state. As both intermediates require stabilization of weak metal-ligand interactions, we surmise the existence of a local structure within the unfolded protein that protects and limits the movement of the ligands, similar to the entatic state found in the native cyt c fold. Furthermore, we observe that in some of the unfolded ensemble, the local stabilizing structure is lost, leading to expansion of the unfolded protein structure and misligation to His26/His33 residues.

Original languageEnglish (US)
Pages (from-to)9788-9800
Number of pages13
JournalChemical Science
Issue number42
StatePublished - 2019

ASJC Scopus subject areas

  • Chemistry(all)


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