Protein Engineering of Electron Transfer Components from Electroactive Geobacter Bacteria

Abstract

Electrogenic microorganisms possess unique redox biological features, being capable of transferring electrons to the cell exterior and converting highly toxic compounds into nonhazardous forms. These microorganisms have led to the development of Microbial Electrochemical Technologies (METs), which include applications in the fields of bioremediation and bioenergy production. The optimization of these technologies involves efforts from several different disciplines, ranging from microbiology to materials science. Geobacter bacteria have served as a model for understanding the mechanisms underlying the phenomenon of extracellular electron transfer, which is highly dependent on a multitude of multiheme cytochromes (MCs). MCs are, therefore, logical targets for rational protein engineering to improve the extracellular electron transfer rates of these bacteria. However, the presence of several heme groups complicates the detailed redox characterization of MCs. In this Review, the main characteristics of electroactive Geobacter bacteria, their potential to develop microbial electrochemical technologies and the main features of MCs are initially highlighted. This is followed by a detailed description of the current methodologies that assist the characterization of the functional redox networks in MCs. Finally, it is discussed how this information can be explored to design optimal Geobacter-mutated strains with improved capabilities in METs.

Keywords: Microbial Electrochemical Technologies (METs); Nuclear Magnetic Resonance (NMR); bioenergy production; bioremediation; electroactive microorganisms; multiheme cytochromes; protein engineering; redox characterization.

Figure 1

Geobacter-based bioremediation technologies and current model for EET in G. sulfurreducens. A conceptual bioremediation station installed near soils, sediments and groundwater contaminated by industrial activities is represented. In this station, organic carbon compounds are introduced to native microorganisms, such as G. sulfurreducens, through injection wells (see [28]). This bacterium couples the oxidation of those compounds with the reduction of different toxic or radioactive compounds, leading to their precipitation and thus facilitating their removal. On the right panel, a model for the EET pathways of G. sulfurreducens is presented. The different proteins that play a role in EET spread from the inner membrane (CbcL and ImcH in orange), through the periplasm (PpcA-family in pink), and to the outer membrane (porin-cytochrome complexes in green). Heme groups are represented in white.

Figure 2

Structures of different c-type cytochromes from Geobacter sulfurreducens obtained in the oxidized state. Monoheme OmcF (PDB ID: 3CU4 [55]), triheme PpcA (lowest energy, PDB ID: 2MZ9 [56]) and dodecaheme GSU1996 (PDB ID: 3OV0 [57]). In all structures, the heme groups are represented in red.

Figure 3

Redox features of monoheme and triheme cytochromes. (A) Electronic distribution scheme of a monoheme (left) and a triheme (right) cytochrome. The inner hexagons represent heme groups, which can either be reduced (colored hexagons) or oxidized (white hexagons). The microstates are grouped according to the number of oxidized hemes in each oxidation stage, connected by consecutive one-electron redox steps. P₀ indicates the fully reduced state. P_ijk indicates the microstates where hemes i, j and k are oxidized. (B) UV-visible spectra of a low-spin c-type cytochrome in the fully oxidized (solid line) and fully reduced (dashed line) states. The inset shows the α-band region of the visible spectra in the redox titration of PpcA [75]. (C) Redox titrations followed by visible spectroscopy of a monoheme (OmcF [55], left panel) and a triheme cytochrome (PpcA [75], right panel). Solid lines indicate the result of the fits for the Nernst equation (OmcF, see text) and for a model of three consecutive reversible redox steps between the different oxidation stages (PpcA, see text). The dashed line in the PpcA panel represents a standard n = 1 Nernst curve with an E_app of −117 mV, illustrating the non-Nernstian redox behavior of a MC.

Figure 4

Ring-current and paramagnetic effects of the heme groups. (A) Model for the ring-current effects in a c-type heme. Based on this model, the chemical shifts of the heme substituents will be affected depending on their location in relation to the heme plane. Consequently, methines (CH), methylenes (CH₂) and methyls (CH₃), whose resonances are usually located between 0.9 and 2 ppm, are found in a wide range of chemical shifts, varying from −1 to 10 ppm (see below). (B) Paramagnetic effects in a triheme cytochrome. The paramagnetic effects (represented by red spheres) caused by the unpaired electrons do not have a linear effect on the chemical shifts of the heme substituents, which can be shifted to lower or higher frequencies on the ¹H NMR spectrum of the cytochrome.

Figure 5

1D ¹H-NMR spectra of the triheme cytochrome PpcA and a diagram of heme c. The typical regions of the hemes substituents in both redox states are indicated. In the heme c diagram, the heme substituents are numbered according to the IUPAC-IUB nomenclature [83]. Dashed and solid lines indicate the connectivities observed in NOESY and TOCSY spectra, respectively. This figure was partially reproduced with the permission of Elsevier from the original work [72].

Figure 6

2D ¹H,¹³C-HSQC NMR spectra of unlabeled (blue contours) and ¹³C labeled (black contours) PpcA obtained at 25 °C, with 640 and 80 scans, respectively. To not overcrowd the figure, only the resonances separated from the main signal envelope are indicated. Blue and black labels indicate the heme substituents and the polypeptide resonances, respectively. The peaks of the protons connected to the same carbon atom (CH₂ groups) are linked by a straight line. In triheme cytochromes c₇, the hemes are numbered I, III and IV [87]. This figure was reproduced with the permission of Elsevier from the original work [72].

Figure 7

Illustration of the heme oxidation profiles of PpcA (pH 8, 15 °C). In the expansions of the 2D ¹H-EXSY NMR spectra, the cross-peaks resulting from intermolecular electron transfer between the different oxidation stages (0–3) are indicated by dashed lines. 1D ¹H-NMR spectra, acquired at different stages of oxidation and illustrating the redox titration of the cytochrome, are represented on top. The peaks corresponding to the heme methyls 12¹CH₃^I, 7¹CH₃^III and 12¹CH₃^IV are marked by green, orange and blue circles, respectively. These heme methyls are also highlighted with the same color code in the heme core of oxidized PpcA (PDB ID: 2MZ9 [56]). This figure was partially reproduced with the permission of Elsevier from the original work [75].

Figure 8

Thermodynamic model for a triheme cytochrome with one redox-Bohr center. (A) Electronic distribution scheme for a triheme cytochrome with a proton-linked equilibrium, showing the 16 possible microstates. The light gray and dark gray circles correspond to the deprotonated and protonated microstates, respectively. The protonated microstates are also identified with a red “H,” which mimics the redox-Bohr center. The reduced hemes i, j and k are colored green, orange and blue, respectively. The oxidized hemes are colored white. P_0H and P₀ represent the reduced protonated and deprotonated microstates, respectively. P_ijkH and P_ijk indicate, respectively, the protonated and deprotonated microstates, where i, j and k represent the heme(s) that are oxidized in that particular microstate. (B) Schematic representation of the interaction networks of a triheme cytochrome [inner hexagons, i (green), j (orange) and k (blue)] and one redox-Bohr center (red ‘H’). The terms g_ij and g_iH represent the interaction energies between the hemes (ij) and between the hemes and the redox-Bohr center (H), respectively. The individual heme oxidation energies are represented as g_i, g_j and g_k for hemes i, j and k, respectively. This figure was partially reproduced with the permission of Portland Press from the original work [89].

Figure 9

Redox titrations followed by visible spectroscopy for PpcA. The redox curves, determined at pH 7 (○) and pH 8 (□), are shown for the reductive (open symbols) and oxidative (filled symbols) titrations. The E_app and $E_1^0–E_3^0$ values result from the fitting of the experimental results to Equation (7) (solid line). This figure was partially reproduced with the permission of Elsevier from the original work [93].

Figure 10

The fitting of the thermodynamic model to the experimental data for PpcA. The solid lines are the result of the simultaneous fitting of NMR and visible data. The figure shows the pH dependence of the heme methyl chemical shifts at oxidation stages 1 (Δ), 2 (□) and 3 (○), and the reduced fractions of the cytochrome, determined by visible spectroscopy at pH 7 (○) and pH 8 (□). The chemical shift dependence of the heme methyl groups in the fully reduced stage (stage 0) is not plotted since they are unaffected by the pH. In the UV-visible redox titration panel, the open and filled symbols represent the data points in the reductive and oxidative titrations, respectively. The experimental uncertainty of the NMR data is evaluated from the line width of each NMR signal at half-height, whereas the uncertainty of the UV-visible data points is usually estimated to be 3% of the total optical signal. This figure was partially reproduced with the permission of Elsevier from the original work [75].

Figure 11

PpcA individual heme oxidation fractions and molar fractions of the 16 individual microstates at pH 7.5. (A) Redox dependence of the heme oxidation fractions of PpcA. The curves for hemes I, III and IV are represented in green, orange and blue, respectively. The oxidation order of the hemes is indicated. (B) Redox dependence of the molar fractions of the 16 microstates of PpcA. Solid and dashed lines indicate the protonated and deprotonated microstates, respectively. For clarity, only the dominant microstates are labeled. In both panels, the curves were calculated as a function of the solution reduction potential (relative to the NHE) using the parameters listed in Table 2. This figure was partially reproduced with the permission of Elsevier from the original work [75].

Figure 12

The spatial location of the residues mutated in the PpcA solution structure. The PpcA polypeptide chain (PDB code: 2MZ9 [56]) is shown as a Cα ribbon in gray, with the heme groups in red. The side chains of V13 and F15 are represented in green; K43, K52 and K60 in blue; and M58 in yellow, all in stick drawings.

Figure 13

Midpoint reduction potentials (E_app) of PpcA mutants. The mutants that retain the e⁻/H⁺ transfer capability are labeled in green, while mutants for which the preferential pathway is disrupted are labeled in red. Wild-type PpcA is labeled in gray.

Figure 14

A schematic representation of the preparation of G. sulfurreducens strains with mutated cytochromes. After the bacterial genome is extracted, the specific target gene is amplified and further inserted into a complementation plasmid. The gene is mutated in key residues through site-directed mutagenesis, and the resultant mutants are inserted into a bacterial knock-out strain. The EET capabilities of the engineered bacteria are then tested in media with different electron acceptors. This process is applied to different key EET components of the bacterium, and the optimized mutants are selected and conjugated. The resultant strain, with higher electron transfer driving force and current production, is finally applied in METs.