Generating single metalloprotein crystals in well-defined redox states: electrochemical control combined with infrared imaging of a NiFe hydrogenase crystal

Abstract

We manipulate and verify the redox state of single metalloprotein crystals by combining electrochemical control with synchrotron infrared microspectroscopy.

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

“…An adaptation of our previously-reported cell design 14,54 was used for single-crystal microspectroscopic electrochemistry, and is described in more detail in the Supplementary Methods (Figure S1). The microspectroscopic-electrochemical cell contained a miniature Ag/AgCl reference electrode (3 M KCl, 2 mm diameter, eDAQ), a graphite ring counter electrode (cut from a graphite tube, Goodfellow), and a glassy carbon working electrode (4 mm diameter, Alfa Aesar).…”

“…[11][12][13] We have previously demonstrated the possibility of electrochemical control over single crystals of hydrogenase I from Escherichia coli (Hyd1) coupled with synchrotron infrared (IR) microspectroscopy for simultaneous reporting on the active site speciation. 14 Vibrational absorption bands of the integral CO and CN  ligands at the active site of hydrogenases make this spectroscopic method ideal for elucidating the redox and coordination state of the active site. By applying steps in electrode potential, we were able to achieve uniform and reversible manipulation of Hyd1 in crystallo from the most oxidised to the most reduced levels.…”

Unifying Activity, Structure, and Spectroscopy of [NiFe] Hydrogenases: Combining Techniques To Clarify Mechanistic Understanding

“…12 Use of high-brightness synchrotron and laser sources allows microspectroscopic IR sampling, lowers the amount of protein required, and enables high signal-to-noise kinetic studies. [13][14][15] [NiFe] hydrogenase structure and mechanism…”

“…(A) Spectra recorded as a function of potential demonstrate accumulation of all active site states in crystallo. (B)Difference spectra recorded as a function of time after a reducing potential step resolve different time dependencies for formation of two Ni a -R sub-states; reproduced with permission from Ash et al14…”

Inhibition in multicopper oxidases: a critical review

“…293 Ash et al presented a route to studying metalloprotein crystals in a defined oxidation state by coupling them to an electrode through redox mediators. 294 This technique, demonstrated with a [NiFe] hydrogenase and coupled to infrared spectroscopic measurements could address the limitations of oxidation state and catalytic form, and could be applied to the study of MCOs.…”