Darren Hsu

Darren is a 2nd year Chemistry student in the Chen lab. He received a BS in Chemistry from National Taiwan University. Darren can be contacted via email here.


Probing Metal Binding Sites and Conformations of Cytochrome c during its Folding Triggered by Electron Transfer

Cytochrome c (cyt c) is a small, soluble hemoprotein that acts as a physiologically ubiquitous electron shuttle. Its folding can be induced from a chemically denatured state via reduction of the heme iron by electron transfer (ET), which leads to changes in the coordination geometry and the free energy of folding which couple to global structural dynamics. Early experiments have shown that the formation time constant of the intermediate states is about 40 microseconds. Multiple candidates including water, His26, His33, and Met65 may bind to the heme iron axially before the native His18 and Met80 ligands. Pentaligated species may also exist. Questions regarding whether the structural changes accompany the initial reduction of Fe(III), if Fe(II) has any additional ligation when the protein starts folding, and whether this ligation is retained during the whole folding process and serves as a nucleation in the protein folding, remain unanswered.

To probe the system, various tools can be employed. The different ligation states can be monitored by X-ray Transient Absorption (XTA) difference spectra through changes in XANES features. Distances between Fe and ligands can be traced by EXAFS. Adopting these non-native ligations leads to different protein ternary structures, resulting in different scattering patterns and radii of gyration. However, previous X-ray Transient Scattering (XTS) studies using continuous flow mixing have been limited to timescales longer than 160 microseconds, and were thus unable to resolve short lived intermediates that occur within the dead time. By incorporating a photo-inducible reactant Ru(bpy)3, which transfers its electron to the heme and initiate the folding process within one microsecond, the intermediate states can be investigated. With sufficient time-resolution and X-ray flux provided by Advanced Photon Source (APS) at Argonne National Laboratory, we seek to complete the description of the ET triggered folding process of cyt c.


Recent Photos

September 12, 2016