Ultrafast Light-Driven Electron Transfer in a Ru(II)tris(bipyridine)-Labeled Multiheme Cytochrome

Abstract

Multiheme cytochromes attract much attention for their electron transport properties. These proteins conduct electrons across bacterial cell walls and along extracellular filaments and when purified can serve as bionanoelectronic junctions. Thus, it is important and necessary to identify and understand the factors governing electron transfer in this family of proteins. To this end we have used ultrafast transient absorbance spectroscopy, to define heme−heme electron transfer dynamics in the representative multiheme cytochrome STC from Shewanella oneidensis in aqueous solution. STC was photosensitized by site-selective labeling with a Ru(II)(bipyridine)3 dye and the dynamics of light-driven electron transfer described by a kinetic model corroborated by molecular dynamics simulation and density functional theory calculations. With the dye attached adjacent to STC Heme IV, a rate constant of 87 × 10 s−1 was resolved for Heme IV → Heme III electron transfer. With the dye attached adjacent to STC Heme I, at the opposite terminus of the tetraheme chain, a rate constant of 125 × 10 s−1 was defined for Heme I → Heme II electron transfer. These rates are an order of magnitude faster than previously computed values for unlabeled STC. The Heme III/IV and I/II pairs exemplify the T-shaped heme packing arrangement, prevalent in multiheme cytochromes, whereby the adjacent porphyrin rings lie at 90° with edge−edge (Fe−Fe) distances of ∼6 (11) Å. The results are significant in demonstrating the opportunities for pump−probe spectroscopies to resolve interheme electron transfer in Ru-labeled multiheme cytochromes. ■ INTRODUCTION Species of Shewanella attract much interest for their ability to respire in the absence of oxygen by transferring electrons from intracellular oxidation of organic matter to extracellular acceptors including Fe2O3 and MnO2 nanoparticles. 1,2 Multiheme cytochromes are essential to this process, and these fascinating proteins are spanned by chains of close-packed ctype hemes. Intraand intercytochrome electron transfer occurs by complementary Fe(III) ↔ Fe(II) transitions of neighboring sites and in this way electrons are moved from the inner bacterial membrane, across the periplasm and outer membrane lipid bilayer to reach the cell exterior. Multiheme cytochromes also contribute to the conductivity of extracellular structures, often termed bacterial nanowires, which transfer electrons across distances greatly exceeding cellular dimensions. These structures for Shewanella oneidensis are multiheme cytochrome containing extensions of the bacterial outer membrane and for Geobacter sulfurreducens are filaments comprised of a polymerized multiheme cytochrome. Beyond their biological role, the remarkable electron transfer properties of multiheme cytochromes have stimulated interest in these proteins as novel bioelectronic junctions and devices. Furthermore, these proteins underpin the wiring of bacteria to electrodes to produce electricity in mediator-less microbial fuel cells and valued chemicals by microbial electrosynthesis. It is now important to identify the factors governing electron transfer in this family of proteins to both understand biology and inspire advances in new, and yet to be conceived, biotechnology. Received: June 28, 2019 Published: August 27, 2019 Article pubs.acs.org/JACS Cite This: J. Am. Chem. Soc. 2019, 141, 15190−15200 © 2019 American Chemical Society 15190 DOI: 10.1021/jacs.9b06858 J. Am. Chem. Soc. 2019, 141, 15190−15200 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. D ow nl oa de d vi a U N IV O F E A ST A N G L IA o n O ct ob er 4 , 2 01 9 at 1 0: 10 :5 1 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s. Multiheme cytochromes are defined by the presence of close-packed c-type hemes, typically with His/His axial ligation, arranged in similar configurations despite very different amino acid sequences and protein folds. Two heme-packing motifs, namely T-shaped and stacked, predominate in the structures resolved to date. Both motifs are present in the periplasmic cytochrome STC from S. oneidensis that is spanned by a chain of four His/His ligated hemes, Figure 1. Heme pairs I/II and III/IV exemplify the T-shaped geometry of neighbors with perpendicular porphyrin rings and edge− edge (Fe−Fe) distances of 5−8 (11−12) Å. The STC heme II/III pair exemplifies the stacked packing motif with parallel porphyrin rings in van der Waals contact and a shorter edge− edge (Fe−Fe) distance of ∼4 (∼9) Å. The possibility that these geometries are optimized to impose control over electron transfer rates and direction has been explored at a singleprotein level through quantum chemistry and molecular simulation. However, to the best of our knowledge, direct measurements of heme−heme electron transfer rates have yet to be reported for STC or other multiheme cytochromes. As a consequence we were motivated to establish whether pump− probe spectroscopy could provide experimental insight into STC heme−heme electron transfer dynamics and, in turn, inform discussions surrounding the mechanism of electron transfer in multiheme cytochromes. Pump−probe spectroscopies, through appropriate combinations of light-triggered electron transfer and time-resolved spectroscopy, offer a powerful way to resolve pathways and dynamics of protein electron transfer across time scales ranging from picoto milli-seconds. The heme−heme electron transfer rate constants in solvated STC are calculated to range from ∼0.5−200 × 10 s−1 and in previous work we established that STC could be labeled site-selectively with [Ru(II)(4-bromomethyl-4′-methylbipyridine) (bipyridine)2] , a thiol-reactive phototrigger of electron transfer. Following photoexcitation into the Ru-dye metalto-ligand charge transfer (MLCT) band, the triplet excited state was oxidatively quenched by heme reduction. Such electron transfer, Scheme 1, pathway 1, (Em Ru(III)/(II*) ≈ −870 mV vs SHE), produces a charge separated state, Ru+:STC−, that will return to the Ru:STC ground state by charge recombination, Scheme 1, pathway 2, (Em Ru(III)/(II) ≈ 1270 mV). However, heme−heme electron transfer in Ru+:STC− could result in each heme existing transiently as Fe(II); the corresponding microscopic Em values 28 lie between −120 and −215 mV as summarized in Figure 1. Gaining direct spectroscopic evidence for electron transfer along the heme wire will be challenging due to the chemical similarity of the hemes. However, we reasoned that heme−heme electron transfer will influence the dynamics of the corresponding photocycle in a manner that could be resolved by ultrafast pump−probe spectroscopy given the time scales predicted for heme−heme electron transfer and lack of protein superstructure, that will inevitably place the Ru(II)-dye in close proximity to the acceptor heme leading to fast charge separation and recombination rates. Here we present ultrafast transient absorbance (TA) of STC proteins photosensitized to inject an electron into opposite ends of the tetraheme chain, into either Heme I or Heme IV as illustrated in Figure 1. Kinetic modeling of the electron transfer dynamics, corroborated by molecular dynamics (MD) simulations and density functional theory (DFT) calculations that provide a microscopic view of the contributing processes, allows us to present rate constants for Heme I ↔ Heme II and Heme IV ↔ Heme III electron transfer that are indicative of fast heme-to-heme electron transfer on the 10 ns time scale. The results are significant in demonstrating the opportunities for pump−probe spectroscopies to resolve interheme electron transfer in Ru-labeled multiheme cytochromes. ■ EXPERIMENTAL METHODS Sample Details. Ru(II)(4-bromomethyl-4′-methylbipyridine) (bipyridine)2(PF6)2 (HetCat Switzerland) was prepared as previously described. All other reagents were analytical grade and aqueous solutions prepared with water having resistivity >18 MΩ cm. Preparation of the STC variants A10C, T23C and S77C and of their photosensitized forms, here termed Ru10:STC, Ru23:STC, and Ru77:STC respectively, was as previously described 9 and outlined in the Supporting Information. Protein concentrations were defined by electronic absorbance of the tetra-Fe(III) forms using ε407nm = 422 mM−1 cm−1 or ε552nm = 29.1 mM −1 cm−1 as reported by Leys et al. TA measurements were performed with anaerobic solutions containing 20 mM TRIS-HCl, 0.1 M NaCl at pH 8.5 and in the absence of sacrificial redox partners. All hemes were in the oxidized, i.e. Fe(III) state, prior to irradiation. Measurements were performed at two protein concentrations to ensure equally good signal-to-noise ratios for quantitative analysis at each wavelength of interest; ∼20 μM protein for 369 and 419 nm and ∼160 μM protein for the less intense features at 453 and 552 nm. The weight-average molecular mass and oligomeric state of Ru77:STC in solutions of 20 mM TRIS-HCl, 0.1 M NaCl at pH 8.5 was defined by analytical ultracentrifugation, sedimentation equilibrium analysis, using a Beckman Optima XL-1 analytical ultracentrifuge equipped with scanning absorbance optics and a Ti50 rotor. Analytical gel filtration was performed with a Superose 6 Increase 10/300 column (GE Healthcare). Time-Resolved Multiple-Probe Spectroscopy (TRMPS). TRMPS TA was performed at the Central Laser Facility of the Figure 1. STC of S. oneidensis illustrating the four hemes (red) and their microscopic reduction potentials in the all-oxidized protein. The Cα atoms of residues changed to Cys for labeling with a Ru(II)dye photosensitizer are indicated as spheres: residues 10 (blue), 23 (yellow), and 77 (cyan). Scheme 1 Journal of the American Chemical Society Article DOI: 10.1021/jacs.9b06858 J. Am.