Tryptophan-accelerated electron flow across a protein-protein interface
We report a new metallolabeled blue copper protein, Re126W122CuI Pseudomonas aeruginosa azurin, which has three redo sites at well-defined distances in the protein fold: ReI(CO)3(4,7-dimethyl-1,10-phenanthroline) covalently bound at H126, a Cu center, and an indole side chain W122 situated between the Re and Cu sites (Re-W122(indole) = 13.1 \xc3\x85; dmp-W122(indole) = 10.0 \xc3\x85, Re-Cu = 25.6 \xc3\x85). Near-UV excitation of the Re chromophore leads to prompt CuI oxidation (<50 ns), followed by slow back ET to regenerate CuI and ground-state ReI with biexponential kinetics, 220 ns and 6 \xce\xbcs. From spectroscopic measurements of kinetics and relative ET yields at different concentrations, it is likely that the photoinduced ET reactions occur in protein dimers, (Re126W122CuI)2, and that the forward ET is accelerated by intermolecular electron hopping through the interfacial tryptophan: *Re//\xe2\x86\x90W122\xe2\x86\x90CuI, where // denotes a protein-protein interface. Solution mass spectrometry confirms a broad oligomer distribution with prevalent monomers and dimers, and the crystal structure of the CuII form shows two Re126W122CuII molecules oriented such that redox cofactors Re(dmp) and W122-indole on different protein molecules are located at the interface at much shorter intermolecular distances (Re-W122(indole) = 6.9 \xc3\x85, dmp-W122(indole) = 3.5 \xc3\x85, and Re-Cu = 14.0 \xc3\x85) than within single protein folds. Whereas forward ET is accelerated by hopping through W122, BET is retarded by a space jump at the interface that lacks specific interactions or water molecules. These findings on interfacial electron hopping in (Re126W122CuI)2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes.
Introduction<!>Materials and sample preparation<!>Protein crystallography<!>Spectroscopic techniques<!>Sample handling<!>Photoinduced electron transfer<!>Solution behavior of Re126W122CuII<!>Crystal structure of Re126W122CuII<!>Intermolecular ET: electronic coupling in (Re126W122CuI)2 and (Re126W122CuII)2<!>Concluding remarks
<p>Electron transfer (ET) between metalloproteins is a fundamental step in biological processes such as photosynthesis and respiration.1 Interprotein ET reactions, which usually occur on μs to ms timescales, can be controlled by gating events that involve exploration of energy landscapes to find productive conformations. A case in point involves dynamic docking of cytochrome b5 with Zn-myoglobin (Mb), Zn-α-hemoglobin,2-6 or Zn-cytochrome c peroxidase,6,7 where a FeIII-heme is reduced by a photogenerated Zn(porphyrin) triplet, *3Zn. Dynamic docking allows a redox protein to find the proper reaction partner and maintain electron flow at a rate commensurate with the final substrate transformation.1</p><p>Much faster (ps-ns) interprotein ET takes place in tightly bound complexes, represented by photosystems I and II of bacterial and plant photosynthesis, where the chlorophyll special pair and nearby redox cofactors are organized in ET active configurations that are fixed in a membrane. This arrangement allows for charge separation in a few ultrafast steps with high conversion efficiency.8 Similar behavior was demonstrated in an artificial system that featured redesign of the cytochrome b5//Zn-myoglobin interface by a triple mutation at the Mb surface, thereby enabling formation of a tight complex with two porphyrins near each other.9-12 Intermolecular ET in the redesigned cytochrome b5//Zn-myoglobin complex was found to be ultrafast, as it occurs from the short-lived (4 ns) singlet state, *1Zn: reaction times for photoinduced *1ZnMb→FeIIIcyt b5 ET and the corresponding back electron transfer (BET) were 400 and 24 ps, respectively, with a rate distribution indicating that the photocycle involves a set of reacting configurations.13 Simulations of this system14 revealed that Mb surface mutations sharply increase the probability of attaining configurations with short distances between cofactors, where the strongest coupled ET pathways involve direct tunneling between the hemes. This finding is in accord with theoretical work on cyt b5 self-exchange, which showed that interprotein ET at short distances could be mediated solely by direct cofactor contact, with no involvement of interfacial water molecules in the dominant ET pathways.15</p><p>Pseudomonas aeruginosa azurin (Az) is a high-potential blue copper protein1 capable of fast and reversible switching between the CuII and CuI oxidation states. Its β-barrel fold is very stable, and its structure is retained upon reducing, removing, or exchanging the CuII atom, modifying the metal binding site16,17 or mutating amino acids in the peptide chain. The combination of fast redox cycling with synthetic flexibility makes azurins promising active components of molecular devices (biomemories or rectifiers).18-24 Photoactive azurin mutants can be prepared by appending Re(CO)3(diimine) photosensitizers to single surface exposed histidine residues and reducing CuII to CuI. Upon near-UV excitation, the metallolabeled proteins can undergo long-range ET from CuI to the electronically excited ReI complex (*Re), with the kinetics dependent on the length and nature of the ET pathways.25-30 Single-step ET between *Re and CuI ceases to be competitive with the ∼1 μs *Re decay as the Cu-Re separation increases. Thus, photoinduced ET was not observed for ReI(CO)3(dmp)H124X122AzCuI (dmp = 4,7-dimethyl-1,10-phenanthroline, H = histidine, X = lysine (K), phenylanine (F), or tyrosine (Y)), where all other native tryptophan and tyrosine residues were replaced with phenylalanine (All-Phe) and the Re and Cu redox centers are separated by 19.4 Å.25 ET was found to be (ultra)fast upon inserting tryptophan (X=W122) into the ET pathway, enabling two-step electron hopping (sequential tunneling) through a W122 intermediate (Scheme 1).25 Electronic coupling in the reactive charge transfer (CT) state is enhanced by delocalization between the dmp ligand of the Re chromophore and the π-interacting W122(indole), facilitating the primary charge separation step.26 (Ultra)fast photoreduction of the Re label was thus observed26 also in the CuII form, in which the CuI/II couple does not participate in the photocycle (Scheme 1).</p><p>We have investigated a new Re-azurin derivative, ReI(CO)3(dmp)H126W122 Az All-Phe (abbreviated Re126W122CuII or Re126W122CuI), where the Re site is two amino acid residues farther away from both W122 and Cu than in the Re124 species, effectively shutting down any direct interaction between the Re(dmp) chromophore and W122. Contrary to our expectations, phototriggered electron flow in Re126W122CuI is as fast as in Re124W122CuI, owing to the presence of a dimer, (Re126W122CuI)2, whose structure features a well-coupled intermolecular ET pathway. We report here a rare case of ET acceleration attributable to hopping across a structurally characterized hydrophobic protein-protein interface.</p><!><p>Site-directed mutagenesis K122W/T126H of plasmid Pseudomonas aeruginosa Az All-Phe H83Q/Y72F/Y108F/W48F was carried out in two addition cycles using a QuikChange kit (Stratagene) with primers purchased from Invitrogen. Plasmids containing the desired mutant were expressed in BL21(DE3) E. coli cells. Protein was collected, purified, and labeled with the Re complex using previously published protocols.31 150 μM [Re(H2O)(CO)3(dmp)](OTf) and 100 μM protein in HEPES buffer were incubated together for 3-4 days at 37 °C and then exchanged into NaPi buffer. Labeled protein was isolated from unlabeled protein using an IMAC copper-chelating column and purified with a Mono Q anion exchange column. Electrospray ionization mass spectrometry of the final product showed a single peak corresponding to Re126W122CuII. The same procedure was used to prepare and characterize Re126K122CuII and Re126F122CuII.</p><!><p>Crystals were obtained by sitting-drop vapor diffusion. Re126W122CuII (3 mg/mL in solution of 40 mM imidazole buffer at pH 7.2 with 2 mM NaCl) was mixed with well solution (100 mM imidazole, 100 mM LiNO3, 6.25 mM CuCl2, 25-30% PEG 4000 at pH 7.0-8.0) in a 1:1 ratio (vol/vol). After 6 months, seed crystals were harvested from two wells (pH 7.4, PEG 27% & pH 7.6, PEG 27%) and used to grow larger crystals over a 4 month period. Crystals were then harvested, coated with a cryoprotectant, and cryogenically stored until data collection. X-ray diffraction data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) Beamline 12-2 on a DECTRIS Pilatus 6M detector and processed with XDS32 and Scala.33 The structure was solved by molecular replacement with Phaser,34 using Chain A of PDB 4AZU35 as a starting model. Coot36 and Phenix37 were both used for model building and refinement, with TLS restraints38 applied in the later refinement cycles. Residual density in some parts was modeled as water. Statistics for data collection, refinement, and validation are provided in Table S1, and the atomic coordinates and structure factors have been deposited in the PDB under entry 4K9J. All molecular graphics were generated using PyMOL.39</p><!><p>Laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS)40 experiments were performed as previously described.41 Microdroplets of sample solution (diameter 50 μm, volume 65 pL) were produced at 10 Hz by a piezo-driven droplet generator and introduced into vacuum via differential pumping stages. The droplets were irradiated by synchronized 6 ns, mJ mid-IR pulses from a home-built optical parametric oscillator (OPO) using LiNbO3 crystals pumped by a Nd:YAG laser. The OPO wavelength was tuned to the absorption maximum of water stretching vibrations (∼3 μm). Pre-formed protein ions were laser desorbed/ablated from the liquid to the gas phase, where they were mass-analyzed in a home-built reflectron time-of-flight (TOF) mass spectrometer with a Daly-type ion detector used for very large biomolecules (m/z up to 106). The delay between the laser and high-voltage ion-accelerating pulses was set at 10 μs. Measurements were performed at two different desorption modes, harsh and soft. Only azurin anions were analyzed. For each experiment, 5 μL of sample solution was loaded into the droplet generator and measured within the first 10 minutes.</p><p>Time-resolved fluorescence and visible transient absorption (TA) experiments on diluted samples (10-100 μM) were performed on a nanosecond laser apparatus with an instrument response time of ca. 20 ns. Details of the apparatus, along with signal acquisition methods, have been described.25,42 Samples were excited with 2-4 mJ/pulse of third harmonic light (355 nm) from a Nd:YAG laser (8 ns pulse width, 10 Hz). The probe light for the TA experiments was provided by either a current-pulsed Xe arc lamp (500 nm) or a HeNe laser (632.8 nm). A photomultiplier tube was used for light detection. Data were collected by a LabVIEW program and later processed and fit to multi-exponentials in Matlab. Samples were placed in either a 1 mm or 1 cm pathlength cuvette and stirred with a magnetic bar throughout the experiment. UV/vis spectra of the sample were collected before and after each measurement. Luminescence lifetimes at higher concentrations (0.09-1.5 mM) were determined using a time correlated single photon counting (TCSPC) IBH 5000 U instrument equipped with a cooled Hamamatsu R3809U-50 microchannel plate photomultiplier and an instrument response time of about 100 ps. Samples in a Hellma microcell were excited at 370 nm with an IBH NanoLED-03 diode laser (∼100 ps fwhm, 500 kHz repetition rate). Luminescence decays were measured at 560 nm (luminescence maximum) at magic angle between the excitation and emission polarization directions. Data were collected in the 0-900 ns range (971.5 ps/channel).</p><p>Time-resolved infrared (TRIR) absorption experiments were performed on the ULTRA instrument43 at the STFC Rutherford Appleton Laboratory. A titanium sapphire laser-based regenerative amplifier (Thales) produced 800 nm, ∼50 fs pulses at a 10 kHz repetition rate. The laser output was split into two parts, one of which was either frequency doubled or used to drive an OPA (Light Conversion, TOPAS) equipped with SHG and SFG units to produce a pump beam at 400 or 355 nm, respectively. The second pumped a TOPAS OPA, yielding signal and idler beams that were difference frequency mixed to generate ∼400 cm-1 broad mid-IR probe pulses. An optical delay line was used to introduce a delay between the pump and probe beams, and the mid-IR probe spectrum was recorded at a given time delay using two 128 element HgCdTe detectors (Infrared Associates). Each detector was attached to a spectrograph, which was tuned to a different spectral region with a small region of overlap on each detector to permit acquisition of the full bandwidth. For nano-microsecond measurements, the sample was pumped with 355 nm, 0.7 ns FWHM pulses (AOT, AOT-YVO-20QSP/MOPO), and probed with electronically synchronized 50 fs IR pulses.44 The sample solutions (0.5-1.0 mM) were placed in a round dip ca. 0.75 mm deep, drilled into a CaF2 plate and tightly covered with a polished CaF2 window. The cell was raster-scanned across the area of the dip in two dimensions orthogonal to the direction of propagation of the laser to prevent laser heating and decomposition of the sample. FTIR spectra were measured before and after the experiment to demonstrate sample stability.</p><!><p>Concentrations of the starting Re126W122CuII solutions (ca. 1 mM) were determined by the absorbance at 632 nm, and the samples were diluted to the final concentrations by adding corresponding buffer solutions. CuI samples were prepared under nitrogen atmosphere by adding either sodium dithionite (TRIR, visible TA, luminescence) or ascorbic acid (visible TA, luminescence) in a buffer solution until discoloration of the CuII sample. Unreacted reductant was removed with a PD10 column for the optical experiments, while a small dithionite excess remained in the case of TRIR experiments. CuI samples were further deoxygenated and stored under argon to minimize oxygen contamination in the spectral measurements.</p><!><p>Photoinduced ET reactions of Re126W122CuII and Re126W122CuI were investigated by a combination of time-resolved spectroscopic techniques over a broad range of sample concentrations (10 μM – 1.5 mM). Measurements on CuII proteins decouple the long-range CuI oxidation to investigate the behavior of the Re126W122 moiety alone, while data on CuI species provide information on the complete photocycle. Luminescence measurements at 560 nm report on decay kinetics of the electronically excited ReI label (*Re, 3CT), and TRIR spectra (Figure 1) show bleaching due to ground-state depletion, blue-shifted bands due to *Re (CT), and red-shifted bands due to reduced Re (CS). Together with time-evolution of the corresponding band areas, TRIR provides structural and kinetics information on the behavior of the Re site (Figure 2). TA of Re126W122CuI at 632.8 and 500 nm reports on the formation and subsequent decay of CuII and reduced Re, respectively (Figure 3).</p><p>Kinetics results of spectroscopic experiments are summarized in Table 1. For both Cu oxidation states, the *Re decay exhibits multiexponential kinetics with lifetimes that are shorter than those of mutants containing K122 or F122 instead of W122. TRIR spectra show, in addition to the decaying 3CT bands, a broad band at ∼1887 cm–1 attributable to the reduced Re chromophore in the 3CS state ReI(CO)3(dmp•–)(W122•+) and, for CuI, also in the reaction product (RP, ReI(CO)3(dmp•–)(W122)AzCuII).25,26,45 The 3CS state is partly formed on a (sub)ps time scale and then rises with a ∼7 ns lifetime, followed by a decay whose kinetics depend on the copper oxidation state: 40-55 ns for CuII and 220 ns and 6 μs for CuI. TA of Re126W122CuI shows a ≤50 ns rise of the CuII signal at 632.8 nm, indicating rapid photoxidation of the CuI site followed by biexponential decay whose lifetimes (220 ns and 6 μs) match those of the CS/RP TRIR band area decay as well as the 500 nm TA decay that combines contributions from *Re and CS.46,47 The 220 ns and 6 μs kinetics are therefore attributable to BET from ReI(CO)3(dmp•–) to CuII.</p><p>This behavior is qualitatively similar to the well-established25,26 electron hopping in Re124W122CuI and Re124W122CuII (Scheme 1), but with several important differences suggesting that photoinduced ET in Re126W122CuI and Re126W122CuII occurs intermolecularly in dimers or oligomers: (1) multiexponential 3CT decay contains an unquenched component whose amplitude increases with decreasing concentration; (2) the maximum intensity of the 3CS TRIR band measured relative to the initial CT3 band intensity is smaller than in the case of the Re124W122Cu species, indicating a lower ET yield; (3) CuII formation upon pulsed laser excitation of Re126W122CuI was observed only with protein concentrations >20 μM and its yield increases with concentration; (4) the initial CuII transient absorbance at 632.8 nm is much smaller for Re126W122CuI than Re124W122CuI measured under comparable conditions; (5) subpicosecond photoreduction of the ReI(CO)3(dmp) label, which was observed in both Re126W122CuII and Re126W122CuI forms, can occur only if ReI(CO)3(dmp) and W122(indole) are in contact prior to excitation, which is not possible within a single molecule.</p><p>Based on these observations, we propose that photoinduced ET in (Re126W122CuII)2 and (Re126W122CuI)2 follows a mechanism (Scheme 2) similar to that established25 for Re124W122CuI (Scheme 1). Regardless of the Cu oxidation state, optical excitation of the Re label produces a singlet charge transfer (CT) state, *1ReII(CO)3(dmp•–). A small fraction undergoes sub-ps reduction by the proximal W122(indole) on the other protein molecule to form the charge-separated (CS) state, ReI(CO)3(dmp•–)//(W122•+)AzCu, where // denotes the protein-protein interface. (Optical excitation of a W122(indole)→//dmp transition also is possible, although less likely due to a very low oscillator strength.26) Most of the 1CT population undergoes ∼150 fs intersystem crossing47,55 to the 3CT state, *ReII(CO)3(dmp•–). (The skewed shape of the TRIR CT1 band and its small initial shift from the corresponding ground state position (ca. +12 cm–1) indicate26 that 3CT is not pure Re→dmp 3MLCT (metal to ligand charge transfer) but also involves partial electron transfer from the neighboring W122(indole).) The ReII center in the 3CT state is reduced by intermolecular ET from W122(indole), establishing an equilibrium with the CS state, which also undergoes ReI(dmp•–)→//W122•+ recombination and, in the case of CuI, intramolecular CuI→W122•+ ET, forming the redox product (RP) ReI(CO)3(dmp•–)//W122AzCuII. The (Re126W122CuI)2 photocycle is completed by ReI(CO)3(dmp•–)→//CuII BET. To account for the biexponential BET kinetics, we propose that the initially formed ReI(CO)3(dmp•–)//W122AzCuII (RPα) undergoes BET and simultaneously establishes an equilibrium with another form (RPβ) that either does not undergo BET or does so very slowly. For the equilibrium constant K >> 1, the observed ∼6 μs BET kinetics component arises from slow replenishment of the RPα population. RPβ formation could involve W122•+ deprotonation or fluctuations at the protein–protein interface.</p><p>The proposed mechanism (Scheme 2) was simulated by utilizing coupled linear differential equations for time-dependent concentrations of 3CT, CS, RPα, and RPβ (details in SI). Values for the 3CT decay were determined separately from the unquenched *Re lifetimes measured in dilute solutions. The rate of CuI oxidation was assumed to be the same as that determined25 for Re124W122CuI as CuI→W122•+ ET follows the same intramolecular path as in Re124W122CuI. Identical values of the forward and back rate constants of the RPα/RPβ conversion were set to reflect the approximately equal amplitudes of the two BET kinetics components in the TA and TRIR experiments. With these assumptions, the simulation yields rate constants for the remaining elementary steps (Scheme 2) that agree with the observed kinetics (Table 1).</p><p>The foregoing interpretation of the ET photoreactivity in Re126W122CuII and Re126W122CuI hinges entirely on the presence of dimers. Next, we will examine possible protein-protein interactions of Re126W122CuII in solutions and crystals.</p><!><p>Unlike other metallolabeled azurins, Re126W122CuII aggregates at concentrations ≥ 0.75 mM, forming blue flakes that can be redissolved upon gentle warming and agitation. Aggregation is even more pronounced at low pH, and gel formation has been observed at <0.5 mM at low temperatures. The behavior of Re126W122CuII in solution has been studied by LILBID-MS, a technique able to determine compositions of noncovalent protein complexes and to provide qualitative information on their state in solution,40,56-58 including unlabeled as well as Re-labeled azurins.41 LILBID-MS results on Re126W122CuII that are relevant to ET reactivity and the nature of its protein-protein interactions (Figures 4, S4) are as follows: (1) Re126W122CuII forms a broad oligomer distribution in solutions (observable up to about 11-mer at present conditions), with a prominent abundance of monomers and dimers. Unlike the trimer and larger oligomers, dimers are still present at the lowest investigated concentration (70 μM), at which LILBID mass spectra of other Re-labeled and unlabeled azurins show only monomers;41 (2) peak broadening and a m/z ≈ +6 kDa shift on going from NH4Pi to NaPi buffer indicate protein solvation and association with sodium and phosphate ions; (3) the dimer peak occurs in NaPi buffer at a mass value that is ∼2.9 kDa lower than double the monomer mass, indicating a large protein-protein interface area in the dimer that is deprived of associated buffer ions and solvating water molecules.</p><p>Whereas LILBID-MS provides evidence for dimer formation in solution, the concentration dependence of Re126W122CuII luminescence decay kinetics reveals its photophysical effects. Global fitting of decay profiles from t > 20 ns at different concentrations and samples yielded lifetimes of 48 and 218 ns. The latter lifetime is comparable to those measured for unreactive mutants Re126F122CuII and Re126K122CuII (Table 1) and is thus attributable to unreactive monomer Re126W122CuII. (Shortening of the luminescence lifetime of the Re126W122CuII monomer in comparison with Re126W122CuI (670 ns) is due to energy transfer from *Re to CuII.) The 48 ns kinetics component is absent in the emission decay of the F122 and K122 mutants. Its presence for Re126W122CuII documents an active role of the W122 residue in *Re quenching in the absence of the CuI reducing center. It is attributable to the reactive dimer (Re126W122CuII)2. Accordingly, the relative amplitude of the 48 ns component increases with increasing sample concentration (Figure 5). Its concentration dependence was analyzed using two protein-protein interaction models described in the SI. While both models assume a monomer-dimer equilibrium and *Re quenching in the dimer, they differ in the relative orientations of the two Re126W122CuII folds. In Model 1, the interaction is asymmetric, such that Re(dmp) of one molecule can interact with W122 of the other one but not vice versa, so that excitation of only one of the two Re centers results in the quenching process. In Model 2, the interaction is symmetric and either of the two Re labels will undergo quenching after excitation. Figure 5 shows that Model 2 is more compatible with the concentration dependence of the relative 48 ns luminescence decay amplitude. (Note that further oligomerization, i.e., addition of monomer(s) to the active dimer, does not substantially alter the behavior predicted by Model 2.)</p><!><p>Re126W122CuII crystallizes in the space group F222 with one molecule in the asymmetric unit (Figure 6). The closest distance between the Re(CO)3(dmp) moiety (REQ, atom C3 of the dmp ligand) and W122 indole (C5) is 10.0 Å with an intramolecular Re-Cu distance of 25.6 Å. The closest intramolecular distance between the W122 indole and Cu is 11.0 Å. Examination of interactions between symmetry related molecules reveals an intermolecular distance REQ(C3)-W122' indole (Nε) of 3.5 Å (residues in the opposite subunit are designated by a prime). The dmp ligand of REQ and the indole side chain of W122' are in a T-like orientation; REQ(Re) and Cu' are separated by 14.0 Å, respectively. The indole rings W122 and W122' are nearly coplanar and positioned edge-to-edge at 4.5 Å. Water molecules are located mostly at the outer surface of the dimer, confirming the hydrophobic nature of the protein-protein interface. Although protein-protein analysis by the software PISA59 did not hint at a classical dimerization interface, the orientation of two neighboring Re124W122CuII molecules suggests that the pair can interact in solution through hydrophobic forces as well as indole-dmp donor-acceptor and dispersion interactions.</p><!><p>The crystallographically determined protein-protein interaction pattern and distances indicate possible pathways that favor intermolecular over intramolecular ET. Indeed, all intramolecular ET distances are much longer than intermolecular ones (Figure 6). The lifetimes for both forward and back ET for monomeric Re126W122 can be calculated using the standard expression for nonadiabatic ET (eq. 1) and were found to be too long in comparison with the experimental data, regardless of the particular mechanism (tunneling or hopping). The rate of intramolecular *Re←CuI ET in Re126W122CuI is estimated to occur with a lifetime of 6.3 ms in the case of single-step tunneling and ≥320 ns for two step *Re←W122←CuI ET, both much longer than the observed <50 ns CuII formation. (The 320 ns hopping time is estimated25,60 using the 10 Å closest dmp-indole intramolecular distance. Using the shortest Re-indole distance (13 Å) gives 8 μs.) The intramolecular BET lifetime in Re126W122CuI can be estimated from that observed in Re124W122CuI (3 μs)25 and the closest Re(dmp)-Cu distances, 23.8 and 15.3 Å, respectively. The estimated value of 34.5 ms is much longer than the observed lifetimes of 220 ns and 6 μs. Hence, only intermolecular ET will be considered in the following discussion.</p><p>(λ = 0.8 eV, k0 = 1013 s–1, r0 = 3 Å, β = 1.1 Å–1, E°(CuII/I) = 0.3 V vs. NHE, E°(*ReII(dmp•–)/ReI(dmp•–)) = 1.25 V, E°(ReI(dmp•–)/ReI(dmp)) = –1.45 V vs. NHE, λ ≈ ΔG0 assumed for forward ET)25,60,61</p><p>As shown in Scheme 2, (Re126W122CuI)2 undergoes *Re//←W122←CuI ET, completed with ReI(dmp•–)//→CuII BET. The key finding is that intermolecular long-range ET in a protein complex can be accelerated by hopping across a protein-protein interface. Fast intermolecular *Re//←W122 ET is mediated by electronic coupling between aromatic rings of Re(dmp) and W122(indole) belonging to different azurin folds, which both lie in close proximity (3.5 Å) at the hydrophobic interface. Additional coupling could occur through dmp Me groups.47,62,63 As a result of these interactions, interfacial 3CT=CS (i.e., *ReII(dmp•–)//W122=ReI(dmp•–)//W122•+) equilibrium is established in a few ns, which is the crucial step in the photocycle as it stores part of the excitation energy and drives long-range CuI oxidation. The latter step, CuI→W122•+, occurs intramolecularly along the same pathway and with virtually the same rate as in Re124W122CuI.25,26</p><p>In addition to the hopping mechanism, it is reasonable to consider single-step intermolecular *Re//←CuI tunneling, for which a lifetime of 18 ns can be estimated assuming that the intermolecular coupling is as strong as that through the single protein fold (eq. 1, r = 14.0 Å, β(Re-Cu) = 1.1 Å–1). The presence of such a competitive reaction, however, is not compatible with the experimental data, as its inclusion in the kinetics model (and varying its time constant from 18 to 200 ns) predicts the *Re decay to occur much faster than the observed ∼7 and 40 ns. The dominance of the hopping mechanism for Re126W122CuI also is supported by the virtually identical 3CT decay and CS rise kinetics observed for CuI and CuII species. Rates of hopping vs. single-step *Re//←CuI tunneling can be estimated from ET hopping maps (Figure 8),25,60 where the ET time is plotted as a function of the driving force for the first tunneling step (–ΔG1sto(*Re//←W122)) and of the overall tunneling process (–ΔGToto(*Re//←CuI)), with black lines enclosing the region in which two-step tunneling is faster than single-step ET. Calculation using parameters relevant for (Re126W122CuI)2 yields a hopping time of 36 ns, in accord with experiment.</p><p>The map shown in Figure 8-left was calculated with all β values equal to 1.1 Å–1, which means that all couplings, including the intermolecular ones, are assumed to be as strong as that mediated by covalent peptide bonds. ET in such a case occurs close to the borderline separating single-step and hopping regions, with single-step tunneling being slightly faster. This assumption, however, is not reasonable for coupling along the single-step Re-Cu intermolecular pathway that involves a 4.5 Å "space jump" that lacks any apparent mediating interactions (Figure 7). Changing β(Re-Cu) to 1.3 Å–1 to account for weaker coupling modifies the hopping region boundaries in such a way that the value calculated for (Re126W122CuI)2 lies in the hopping region (Figure 8-right). The proximity to the single-step/ hopping boundary suggests a possibility of a mixed process whereby both processes would contribute. Such a situation emerged from theoretical analysis of photoinduced ET in the ZnCcP//Cyt c complex,12 where only a fraction of ET events involves hopping, depending on the particular Trp environment during structural fluctuations. In the case of (Re126W122CuI)2, we can conclude on kinetics grounds that the hopping mechanism is dominant. This conclusion is further supported by the virtually identical nature and kinetics of early ET steps in the CuII form that demonstrate a direct reduction of *Re in the absence of any other reducing site except W122.</p><p>In contrast to the forward ET pathway, the protein-protein interface in (Re126W122CuI)2 is not well coupled for ReI(dmp•–)//→CuII BET (Figure 7). Based on the 3 μs BET in Re124W122CuI over a CuII-dmp•– distance of 15.3 Å,25 the BET lifetime in (Re126W122CuI)2 (11.6 Å) can be estimated (eq. 1) as 51 ns. Both observed BET lifetimes (220 ns, 6 μs) are much slower, suggesting that, despite a short CuII-dmp•– distance, coupling across a protein-protein interface is weaker than that along the peptide chain in Re124W122CuI, most likely attributable to the presence of 3-4 Å "space jumps", absence of intermolecular donor-acceptor and H-bonding interactions along the BET pathway, as well as to the absence of potentially mediating interfacial H2O molecules (Figure 7).</p><p>Although other Re-labeled azurins also form oligomers in concentrated solutions,41 the well-coupled interface is a unique feature of the forward ET in (Re126W122CuII)2. Crystals of All-Phe Re107CuII and Re124K122CuII show two molecules interacting through dmp-dmp (phen-phen in the latter) π-stacking that connects the two Re labels (Figure 9), precluding intermolecular ET due to long Re-//-Cu distances.51 The Re124W122CuII crystal structure (Figure 9) features a W122(indole)-Re(dmp)//Re(dmp)-W(indole) π-stacked motif26 that would enable only intramolecular W122(indole)→*Re(dmp) ET, the interface acting as an insulating barrier. In the case of Re(phen)126K122CuII, the crystal structure shows the Re(CO)3(phen) unit of one azurin molecule positioned against the peptide chain of a neighboring protein, while the Re sites of the two molecules are laterally separated from each other by amino acid side chains of different β-sheets of the two protein folds (Figure 9).51 In all these cases, the azurin-azurin interface contains several water molecules. A single K122W mutation in Re126K122CuII, which introduces the aromatic amino acid tryptophan, produces a dramatically different Re126W122CuII structure (Figure 6) with a large hydrophobic interface and a pair of Re(dmp)-W122(indole) intermolecular contacts.</p><!><p>The protein-protein interaction pattern in (Re126W122CuI)2 and (Re126W122CuII)2 features a well-coupled intermolecular ET hopping pathway, namely Re(dmp)//W122(indole)–Cu, where the distances between photoredox active units of neighboring molecules are much shorter than respective intramolecular ones (Figure 6). The nature of the *ReII(dmp•–)//W122 interfacial interaction is akin to the strongly coupled "direct contact regime" with no bridging water molecules, which emerged from dynamics simulations of cyt b5//cyt b5 self-exchange15 and of the tightly bound Mb-cyt b5 mutant.14 The *ReII(dmp•–)//W122 coupling is strong enough to mediate sub-ps ET from the 1CT state and to establish a redox equlibrium with the 3CS state ReI(dmp•–)//W122•+ on a timescale of a few ns (Scheme 2). Unlike the forward reaction, BET is not accelerated by hopping, since CuII is not able to oxidize tryptophan.</p><p>Acceleration of intermolecular ET by hopping across interfaces in protein complexes is rare in nature. One notable case involves hole transfer between two tyrosine residues (Tyr731//Tyr356) at the boundary between α and β subunits of ribonucleotide reductase, although the nature of the coupling is not known.60,64,65 There are many more examples of intraprotein hopping as documented in photolyases, ribonucleotide reductase, MauG, and in photosystems I and II. Some of these systems were recently analyzed using hopping maps.60,66 The 3-membered "tryptophan wire" in photolyase clearly demonstrates the importance of the right orientation and interaction of indole groups and the chromophore that allow for 30 ps ET across about 15 Å.67</p><p>It is interesting to note that tryptophan acts as a "rectifier", accelerating ET in only one direction. Re-azurins exemplify the case of forward ET acceleration, whereas selective BET acceleration was observed in (Zn-cyt c P)//(cyt c) where FeII-cyt c → Zn-cyt cP+ BET occurs by hopping through W191 that is positioned close to the cyt cP heme but not directly at the protein-protein interface.7,12,68 A single-docking ET mechanism featuring one-way hopping acceleration through tryptophan placed at a well-coupled hydrophobic protein-protein boundary, documented by our work on (Re126W122CuI)2, emerges as a promising strategy to realize long-lived charge separation in protein complexes.</p>