Delineating redox cooperativity in water-soluble and membrane multiheme cytochromes through protein design

Abstract

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

Keywords: de novo protein design; electrostatic calculation; heme proteins; membrane protein design; redox cooperativity.

FIGURE 1

The suite of monoheme and diheme water‐soluble and transmembrane modular redox proteins based on the coiled‐coil diheme protein 4D2. (a) m1‐4D2 contains heme 1 but heme site 2 is re‐designed. (b) 4D2 is the basis of the protein suite and contains two identical bis‐histidine heme‐binding sites. (c) m2‐4D2 contains heme 2 but heme site 1 is re‐designed, containing the same core packing residues as in site 2 of m1‐4D2. (d) m1‐CytbX contains heme 1 of CytbX but heme site 2 was re‐designed with Rosetta. (e) CytbX is the transmembrane version of 4D2. (f) m2‐CytbX contains heme in site 2 but heme site 1 was re‐designed with Rosetta. All structures are Rosetta‐generated models, apart from 4D2 which is the crystal structure (PDB ID: 7AH0) with reconstructed loops. Protein backbones are shown as gray ribbons, hemes (red), mutated core residues (cyan) and heme‐coordinating residues (gray) are shown as gray sticks. The sequence of each protein is shown, with mutated residues highlighted in cyan, histidines highlighted in red, and loop residues in gray.

FIGURE 2

Biophysical characterization of monoheme designs. UV–Vis absorbance spectra of purified (a) m2‐4D2, (b) m1‐CytbX and (c) m2‐CytbX. Reduced spectra are cropped at the point where the detector is saturated by dithionite absorbance. Absorbance is normalized to the oxidized Soret peak maximum. Circular dichroism spectra during thermal melts of purified (d) m2‐4D2, (e) m1‐CytbX and (f) m2‐CytbX from 5°C to 95°C. All designs bind heme, are helical and highly thermostable as designed.

FIGURE 3

Redox potentiometry of the monoheme and diheme (a) water‐soluble proteins and (b) membrane proteins. Measurements were performed with purification tags removed from all proteins. Membrane proteins: PH = 7.4, soluble proteins: PH = 8.6. Buffer for soluble proteins: 20 mM CHES, 100 mM KCL, pH 8.6. Buffer for membrane proteins: 50 mM sodium phosphate (pH 7.4), 150 mM NaCl, 5% glycerol, 0.08% CYMAL‐5. Poisson‐Boltzmann Monte‐Carlo electrostatics calculations of redox titrations for (c) m1‐4D2 vs. m2‐4D2 and (d) m1‐CytbX vs. m2‐CytbX. Water‐soluble proteins were modeled in a solvent with a dielectric constant (ε_p) of 20 at pH 8.6, and membrane proteins were modeled in an implicit membrane with ε_m = 4 at pH 7.4. Predicted E_m shifts are reported using m1‐4D2 and m1‐CytbX as references for m2‐4D2 and m2‐CytbX respectively. Redox potentials were derived by fitting to a Nernst equation describing a single electron transfer.

FIGURE 4

(a) BioDC reproduces the trend in redox potential shifts across the mono‐ and diheme constructs in aqueous and membranous environments, and (b) delineates the contributions to the redox potential splitting in the dihemes in terms of environmental electrostatics and heme‐heme interactions. Hemes in site 1 and 2 of the diheme constructs are denoted by suffixes (e.g. 4D2_1 denotes 4D2 heme 1). Note that the relative redox potentials for the dihemes from experiment were assigned to heme site 1 and 2 based on the BioDC results. The purple diamonds and squares correspond to results obtained using atomic partial charges for the heme derived according to the Restrained Electro‐Static Potential (RESP) and Charge Method 5 (CM5) schemes, respectively.