Designed metalloprotein stabilizes a semiquinone radical

Abstract

Enzymes use binding energy to stabilize their substrates in high-energy states that are otherwise inaccessible at ambient temperature. Here we show that a de novo designed Zn(II) metalloprotein stabilizes a chemically reactive organic radical that is otherwise unstable in aqueous media. The protein binds tightly to and stabilizes the radical semiquinone form of 3,5-di-tert-butylcatechol. Solution NMR spectroscopy in conjunction with molecular dynamics simulations show that the substrate binds in the active site pocket where it is stabilized by metal-ligand interactions as well as by burial of its hydrophobic groups. Spectrochemical redox titrations show that the protein stabilized the semiquinone by reducing the electrochemical midpoint potential for its formation via the one-electron oxidation of the catechol by approximately 400 mV (9 kcal mol(-1)). Therefore, the inherent chemical properties of the radical were changed drastically by harnessing its binding energy to the metalloprotein. This model sets the basis for designed enzymes with radical cofactors to tackle challenging chemistry.

Figure 1. Semiquinone is unstable as a free radical, but its stability can be tailored in a protein environment

a, Scheme of a canonical equilibrium between QH₂, the anion radical SQ^• and Q. The SQ^• anion radical is an intermediate between the fully reduced QH₂ and the fully oxidized Q. The corresponding energy versus reaction coordinate plot is representative of the expected differences in thermodynamic potential for each discrete oxidation state. The red arrows indicate that the environment of SQ^• can change the SQ^• stability by shifting its thermodynamic potential. b, General scheme for reconstituting DFsc with a radical. SQ^• is generated in situ via comproportionation of QH₂ and Q and consumed by disproportionation as a free radical. In the presence of Zn(ii)-bound DFsc ([DFsc-Zn(ii)₂]), the equilibrium is shifted towards the otherwise unstable SQ^•, which complexes with the protein to form [DFsc-Zn(ii)₂]–SQ^•.

Figure 2. Observation of SQ^• in complex with the de novo metalloprotein [DFsc-Zn(ii)₂] by optical and magnetic spectroscopy

a, UV/vis absorption spectra were recorded starting from the time of QH₂ and Q mixing to generate SQ^• in situ (black line), and at ten minute intervals up to 180 minutes (red line) (a.u., arbitrary units). b, EPR spectra of [DFsc-Zn(ii)₂], apo DFsc and Zn(ii) in the presence of SQ^• generated in situ. c, 2D HSQC spectra of [DFsc-Zn(ii)₂] (black) and after 74% of all the protein was converted into [DFsc-Zn(ii)₂]–SQ^• (red). Backbone assignments are labelled and are in good agreement with previous studies. All amide resonances are visible at this contour level except for A90.

Figure 3. Analysis of results extracted from the [DFsc-Zn(ii)₂]–SQ^• HSQC spectra colour-mapped on the [DFsc-Zn(ii)₂] structure (PDB 2LFD), with the relative degrees of peak intensities compared

a,b, The highest degree of reduced peak intensity corresponds to the highest PRE. Colour-mapped structures are from the side (a), and top-down views, with the backbone amide nitrogen atoms displayed as spheres and the loop regions omitted for clarity (b). For both panels, Zn(ii) is shown as grey spheres and Zn(ii)-coordinating residues as sticks. Residues that experience the highest PRE are coloured red on the structural models and below in the corresponding sequence. Residues that coordinate to Zn(ii) are underlined in the sequence. All molecular graphics were generated with PyMOL.

Figure 4. QM/MM-optimized model of [DFsc-Zn(ii)₂]–SQ^•

a, View of the DFsc active site showing the first coordination shell of the di-Zn(ii) (white spheres), with the SQ^• coloured magenta. Dashed lines indicate bonds. b, Top-down view of the [DFsc-Zn(ii)₂]–SQ^• complex showing the enlargement of helix 1 (blue) and helix 2 (green) to accommodate SQ^• binding. c, View of the hydrophobic residues that line the helix 1 and 2 interface (shown as spheres: A10, G14, I17, A43, G47, V50, Y51), which interact with the t-butyl groups of SQ^• (in magenta).