Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF

Abstract

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.

Keywords: density functional theory; electron transfer; extracellular respiration; heme; molecular dynamics.

Fig. 1.

Crystal structures of decaheme cytochromes MtrC (PDB ID code 4LM8; A) (17) and MtrF (PDB ID code 3PMQ; B) (15), pentaheme cytochrome NrfB (PDB ID code 2OZY; C) (13), and tetraheme cytochrome STC (PDB ID code 1M1Q; D) (48). The bis-His coordinated c-type heme rings are depicted in green, Fe atoms are in purple, and the protein secondary structures are in gray. (E) Cartoon representation of a possible arrangement of MtrCAB complexes in the bacterial outer membrane (OM) during extracellular respiration inspired by the cryotomography study in ref. . Electrons from the periplasm are transferred across the OM via the decaheme protein complex MtrAB and passed onto the decaheme protein MtrC, where the electron flow is trifurcated in directions parallel and orthogonal to the OM. The spacing between the centers of adjacent MtrC and MtrA molecules is typically about 10 nm (i.e., close contact), but gaps larger than 30 nm were also observed and may be overcome by lateral protein diffusion within the membrane as indicated by dashed arrows (19). IM, inner bacterial membrane.

Fig. 2.

Heme–heme electronic coupling matrix elements, $\left|H_{\text{ab}}\right|$, in MtrC, MtrF, and STC. The distance dependence of electronic couplings is shown in A for the minimum QM model ($H_{\text{ab}}=H_{\text{ab}}^{\text{m}}$) composed of the unsubstituted heme rings plus axial ligands and in D for the large QM model ($H_{\text{ab}}=H_{\text{ab}}^{\text{l}}$), where, in addition, all heme side chains are included, in particular the Cys linkages. The couplings are calculated on structures obtained from MD simulation at room temperature. They are color coded according to the relative orientations of electron donating and accepting hemes: stacked motif in blue (heme pairs 10–9, 9–8, 3–4, and 4–5 in MtrC and MtrF and 2–3 in STC), T shaped in red (8–6 and 1–3 in MtrC and MtrF and 1–2 and 3–4 in STC), and coplanar in green (6–1, 6–7, and 1–2 in MtrC and MtrF). Root-mean-square averages of the scattered data points were calculated for bins of width 0.4 (A) and 0.2 Å (D) and are denoted by black circles, with error bars indicating the root-mean-square fluctuations. Fits to an exponential are indicated by black lines. In A, the shortest heme edge-to-edge distance is used, and in D, the shortest distance between any heavy atom of heme ring and side chains is used. Electronic couplings averaged for each adjacent heme pair in MtrC, ${⟨{\left|H_{\text{ab}}\right|}^2⟩}^{1/2}$, are indicated for the minimum QM model (B) and for the large QM model (C). The thickness of the bars connecting adjacent hemes is proportional to the average coupling. C, Insets depict the enhancement of electronic couplings due to Cys linkages inserting in the space between coplanar heme pair 6–1 and T-shaped heme pair 8–6. One of the three Fe $d\left(t_{2g}\right)$-heme orbitals on electron donor and acceptor hemes contributing to electronic coupling are drawn as red/yellow and green/blue isosurfaces (denoted $d_i^{\text{D}}$ and $d_j^{\text{A}}$ in SI Appendix). Similar coupling enhancements are found for MtrF.

Fig. 3.

Outer-sphere reorganization free energy, ${\lambda }_{\text{o}}$, for heme-to-heme ET in MtrC (blue), MtrF (green), NrfB (black), and STC (red) as obtained from MD simulations. Values for MtrC and NrfB are taken from present simulations (SI Appendix, Table S1), values for MtrF are taken from ref. , and values for STC are taken from ref. . Correlations are shown between ${\lambda }_{\text{o}}$ and (A) the solvent-accessible SA (49) of corresponding heme pairs or (B) Marcus continuum estimates for outer-sphere reorganization free energy, ${\lambda }_{\text{o}}^{\text{s}}$, with SA-dependent static dielectric constant (details are in the text).

Fig. 4.

Kinetics of trifurcated electron flow in solvated MtrC (A) and MtrF (B). The thickness of the colored arrows connecting hemes is proportional to the heme-to-heme ET rate constants in the all-ox state, which are summarized in SI Appendix, Table S1. Fig. 1 shows heme numbering. Insets show the possible flow directions between the terminal hemes 10, 5, 7, and 2, with the logarithm of the maximum protein-limited, steady-state electron flux, ${\log }_{10}\left(J_{\text{max}}/s^{-1}\right)$, indicated for each flow direction. The electron flux is obtained by solving a chemical Master equation; details are in Materials and Methods and SI Appendix. $J_{\text{max}}$ is taken from SI Appendix, Table S2 (“ox-$\mathrm{s}\mathrm{c}$”).