The porphyrin ring rather than the metal ion dictates long-range electron transport across proteins suggesting coherence-assisted mechanism Yuval Agama, Ramesh Nandia, Alexander Kaushanskya, Uri Peskina, and Nadav Amdurskya,1 aSchulich Faculty of Chemistry, Technion–Israel Institute of Technology, 3200003 Haifa, Israel Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved November 2, 2020 (received for review May 4, 2020) The fundamental biological process of electron transfer (ET) takes distances, has also been observed across the multicellular body of place across proteins with common ET pathways of several nano- cablebacteria(19–21). The only common feature of long-range meters. Recent discoveries push this limit and show long-range extracellular ET is the presence of large multiheme cytochromes. extracellular ET over several micrometers. Here, we aim in deci- The mechanism of this extraordinary ET as well as the nature of phering how protein-bound intramolecular cofactors can facilitate the bacterial nanowire is a source of great scientific debate. The such long-range ET. In contrast to natural systems, our protein- two most frequently discussed ET mechanisms are: 1) incoherent, based platform enables us to modulate important factors associ- nonadiabatic electron hopping across the Fe ions within the heme ated with ET in a facile manner, such as the type of the cofactor (8, 22, 23); and 2) metallic-like or coherent ET across the bacterial and its quantity within the protein. We choose here the biologi- nanowire, perhaps involving an aromatic sidechain of the protein cally relevant protoporphyrin molecule as the electron mediator. (13, 14, 24). Recent electron-beam (e-beam) diffraction studies Unlike natural systems having only Fe-containing protoporphyrins, have revealed the presence of a rather amazing type of protein i.e., heme, as electron mediators, we use here porphyrins with nanowire of the G. sulfurreducens, composed solely of the hexa- different metal centers, or lacking a metal center. We show that heme cytochrome OmcS (25, 26). The existence of this unique the metal redox center has no role in ET and that ET is mediated one-dimensional (1D) wire assembly of heme molecules strongly solely by the conjugated backbone of the molecule. We further suggests that they mediate ET, regardless of the ET mechanism. discuss several ET mechanisms, accounting to our observations CHEMISTRY with possible contribution of coherent processes. Our findings While bacterial mutants may be created in order to study the role contribute to our understanding of the participation of heme mol- of the different proteins participating in the ET pathway, it is nearly ecules in long-range biological ET. impossible to specifically investigate the role of the heme molecules in ET, due to the ubiquity of the iron-containing heme molecule. In G. sulfurreducens electron transfer | conductive polymers | heme | porphyrin | biopolymers thecaseofthe nanowires of the OmcS protein, the coordination with the Fe ion iswhatdrivestheself-assembly process, and, therefore, the iron cannot be removed. ron-containing protoporphyrin, also known as heme, is one of Here, we introduce a completely different approach to the the most important molecules in nature, participating in nu- BIOPHYSICS AND COMPUTATIONAL BIOLOGY I study of very-long-range ET across proteins. We achieve this by merous biological processes, including the binding of gas mole- creating an artificial protein-based fibrillar platform, in which we cules (as O ) to its iron ion. The proper activity of heme 2 can control the number of ET-mediating cofactors inside the pro- molecules is dependent on the ability of specific proteins, which tein, as well as their chemical nature. Inspired by the extraordinary are referred to as hemeproteins, to bind them. The natural ac- tivity of heme is owed to its exceptional heme-iron coordination with gas molecules or specific amino acids. In fact, an iron-free Significance porphyrin or a degraded product of heme, bilirubin, can result in certain diseases, such as porphyria or jaundice, respectively. Other Electron transfer (ET) across proteins is fundamental in many metal-containing protoporphyrins are far from being as ubiquitous biological processes, such as photosynthesis and aerobic res- as the iron-containing one. Although there are many kinds of piration. Recent discoveries from the bacterial world have hemeproteins with different functionalities, such as hemoglobins, shown that proteins can support long-range ET pathways on myoglobins, cytochromes, and peroxidases, we will focus here on the order of micrometers. Here, we introduce a protein plat- their ability to mediate electrons (1). Hemeproteins participate in form that allows us to explore how proteins can support long- all major electron transfer (ET) chain reactions in nature, the range ET, and what are the important molecular features and most noteworthy being the aerobic respiration system in our mi- ET mechanisms that enable it. We focus on the role of the metal ion within heme molecules in long-range ET and find tochondria and the photosynthetic system in chloroplasts. In ET that the molecular ring surrounding the metal ion is the ET reactions, electrons are shuttled by the redox of the Fe ion within mediator, and not the metal ion. We suggest a coherence- the heme (Fe2+ ⇌ Fe3+), whose redox potential is highly de- assisted ET mechanism as a possible explanation for our high pendent on the heme-binding site within the protein (2, 3). measured ET efficiency. Until recently, all of the natural ET pathways studied were on the nanometer scale (usually below 10 nm), and the heme was Author contributions: N.A. designed research; Y.A. and R.N. performed research; A.K. and located at the correct place along the electrochemical gradient, U.P. contributed new theoretical/analytic tools; Y.A., R.N., A.K., U.P., and N.A. analyzed with other cofactors (such as quinones or FeS clusters) that data; and Y.A., U.P., and N.A. wrote the paper. participate in the ET chain reaction as well. Recent examples The authors declare no competing interest. from the bacteria world have shown, however, that specific bacteria This article is a PNAS Direct Submission. (namely, Geobacter sulfurreducens and Shewanella oneidensis MR- Published under the PNAS license. 1) can mediate ET with very high efficiency over very long distances 1To whom correspondence may be addressed. Email: [email protected]. in the order of micrometers, and even up to millimeters, from This article contains supporting information online at https://www.pnas.org/lookup/suppl/ the bacteria’s body to nearby metal-oxide rocks across a bacte- doi:10.1073/pnas.2008741117/-/DCSupplemental. rial nanowire (4–18). Extremely long ET, over centimeter-scale www.pnas.org/cgi/doi/10.1073/pnas.2008741117 PNAS Latest Articles | 1of7 Downloaded by guest on September 27, 2021 extracellular ET observed with OmcS nanowire, we chose here only S3). This result clearly implies that the observed long-range ET protoporphyrin as the electron-mediating cofactors across our across our protein platform is not mediated by the metal ion protein-based platform, although other macromolecules are known within the PPIX and that charge-carrier mobility is enhanced by to participate in various biological pathways. Using our platform, the conjugated π-system of the porphyrin rings themselves. To we can now change the metal ion inside the porphyrin, remove the establish this assumption, we also examined bilirubin as a dop- metal ion altogether, or even use bilirubin (the open form of the ring) ant, since its molecular structure has the same conjugated tet- as ET mediators. We used electrochemical impedance measure- rapyrrole motif as PPIX, albeit linear rather than cyclic. We ments, as well as three-terminal transistor measurements, to explore found that bilirubin indeed exhibited similar improvements in the role of the porphyrin variant in mediating ET. Our results allow conductivity, as was observed for the other porphyrin dopants us to discuss possible mechanisms of ET across our protein platform, (Fig. 2A). To further explore the ET mechanism across the dif- involving either incoherent or coherence-assisted ET. ferent mats, we measured the temperature dependence of the EIS response, displayed in the form of a Nyquist plot (the im- Results pedance imaginary part as a function of the impedance real part) The protein-based macroscopic platform that we use here is (SI Appendix, Fig. S4). The plot shows a semicircle in the high- based on electrospun bovine serum albumin (BSA) mats, which frequency area, corresponding to bulk conductivity, and a curved were shown to be able to bind heme molecules, resulting in long- tail in the low-frequency area, attributed to double-layer capac- range ET across this artificial platform (27). As stated, here, we itance at the electrolyte and blocking-electrode interface, inter- aim to understand how protoporphyrins mediate ET. Accord- rupted by the ionic diffusion process. In these temperature- ingly, we molecularly doped the BSA mats with different vari- dependence studies, we found that all of the doped mats had a ants: metal-free protoporphyrin IX (PPIX), the common Fe- similar activation energy in the order of 0.2 ± 0.03 eV (SI Ap- containing PPIX (heme), PPIX with three other transition pendix, Table S1). The similar activation energy values imply that metals (Co, Cu, and Zn), and bilirubin. The successful doping of all of the mats we used, some of which contained a metal ion the BSA mat is easily observed by a distinct color change of the within the PPIX and some of which did not, share a similar ET mat in accordance with the porphyrin color (Fig. 1). Moreover, mechanism (as discussed below). the doping level, i.e., the number of dopants per volume within After establishing that the conductivity of the doped mats is the mat, can be easily modulated by the duration of the doping determined only by the amount of porphyrin bound to the BSA process and can be verified by using ultraviolet-visible (UV-Vis) mat, regardless of the metallic element (in analogy to semicon- “ ” spectroscopy.