Substitution of the sole tryptophan of the cupredoxin, amicyanin, with 5-hydroxytryptophan alters fluorescence properties and energy transfer to the type 1 copper site.

Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino acid, 5-hydroxytryptophan (5-HTP). The 5-HTP substituted amicyanin exhibited absorbance at 300-320 nm, characteristic of 5-HTP and not seen in native amicyanin. The fluorescence emission maximum in 5-HTP substituted amicyanin is redshifted from 318 nm in native amicyanin to 331 nm and to 348 nm in the unfolded protein. The fluorescence quantum yield of 5-HTP substituted amicyanin mutant was much less than that of native amicyanin. Differences in intrinsic fluorescence are explained by differences in the excited states of tryptophan versus 5-HTP and the intraprotein environment. The substitution of tryptophan with 5-HTP did not affect the visible absorbance and redox potential of the copper, which is 10 Å away. In amicyanin and other cupredoxins, an unexplained quenching of the intrinsic fluorescence by the bound copper is observed. However, the fluorescence of 5-HTP substituted amicyanin is not quenched by the copper. It is shown that the mechanism of quenching in native amicyanin is Förster, or fluorescence, resonance energy transfer (FRET). This does not occur in 5-HTP substituted amicyanin because the fluorescence quantum yield is significantly lower and the red-shift of fluorescence emission maximum decreases overlap with the near UV absorbance of copper. Characterization of the distinct fluorescence properties of 5-HTP relative to tryptophan in amicyanin provides a basis for spectroscopic interrogation of the protein microenvironment using 5-HTP, and long-distance interactions with transition metals.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.

Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans.

F1FO-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.

Efficient total nitrogen removal from wastewater by Paracoccus denitrificans DYTN-1.

Bioaugmentation is an effective treatment method to reduce nitrogenous pollutants from wastewater. A strain of DYTN-1, which could effectively remove TN from sewage, was isolated from the sludge of a wastewater treatment plant and was identified as Paracoccus denitrificans. The TN in wastewater reduced to <20 mg l-1 within 12 h under optimal conditions by free cells of P. denitrificans DYTN-1. To enhance the removal of TN, P. denitrificans DYTN-1 cells were immobilized in sodium alginate (SA) using different divalent metal ions as cross-linking agents. It was found that the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 to below 20 mg l-1 within 8 h. After the optimization of an orthogonal experiment, the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 mg l-1 to below 20 mg l-1 within 1 h and significantly reduce the fermentation cycle. These findings would provide an economical and effective method for the removal of total nitrogen in wastewater by immobilized cells of P. denitrificans DYTN-1. SIGNIFICANCE AND IMPACT OF THE STUDY: We identified a new Paracoccus denitrificans strain (DYTN-1) for removal of the total nitrogen in wastewater. The total nitrogen could be removed effectively by P. denitrificans DYTN-1 within 12 h in wastewater. Using sodium alginate as the carrier and Ba2+ as cross-linking agent, the immobilized P. denitrificans DYTN-1 cells could improve the removal efficiency of total nitrogen in wastewater and significantly reduce the fermentation cycle. The assay has provided an economical and effective method for the removal of total nitrogen in wastewater by immobilized cell.

Arginine-95 is important for recruiting superoxide to the active site of the FerB flavoenzyme of Paracoccus denitrificans.

Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.

Geometrical Description of Protein Structural Motifs.

We present a geometrical method that can identify secondary structural motifs in proteins via angular correlations. The method uses crystal structure coordinates to calculate angular and radial signatures of each residue relative to an external reference point as the number of nearest-neighbor residues increases. We apply our approach to the blue copper protein amicyanin using the copper cofactor as the external reference point. We define a signature termed Δß which describes the change in angular correlation as the span of nearest neighbor residues increases. We find that three turn regions of amicyanin harbor residues with Δß near zero, while residues in other secondary structures have Δß greater than zero: for ß-strands, Δß changes gradually residue by residue along the strand. Extension of our analysis to other blue copper proteins demonstrated that the noted structural trends are general. Importantly, our geometrical description of the folded protein accounts for all forces holding the structure together. Through this analysis, we identified some of the turns in amicyanin as symmetrical anchor points.

O2 versus N2O respiration in a continuous microbial enrichment.

Despite its ecological importance, essential aspects of microbial N2O reduction-such as the effect of O2 availability on the N2O sink capacity of a community-remain unclear. We studied N2O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N2O and O2 can occur, (ii) the mechanism governing the competition for N2O and O2, and (iii) how the N2O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N2O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O2. When supplied simultaneously, N2O reduction to N2 was only detected when the O2 concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N2O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture's affinity constant (KS) for O2 was found to be two orders of magnitude lower than the value for N2O, explaining the preferential use of O2 over N2O under most environmentally relevant conditions.

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics.

The mechanism for oxygen reduction in cytochrome c dependent nitric oxide reductase (cNOR) as obtained from a combination of theoretical and experimental results.

Bacterial NO-reductases (NOR) belong to the heme-copper oxidase (HCuO) superfamily, in which most members are O2-reducing, proton-pumping enzymes. This study is one in a series aiming to elucidate the reaction mechanisms of the HCuOs, including the mechanisms for cellular energy conservation. One approach towards this goal is to compare the mechanisms for the different types of HCuOs, cytochrome c oxidase (CcO) and NOR, reducing the two substrates O2 and NO. Specifically in this study, we describe the mechanism for oxygen reduction in cytochrome c dependent NOR (cNOR). Hybrid density functional calculations were performed on large cluster models of the cNOR binuclear active site. Our results are used, together with published experimental information, to construct a free energy profile for the entire catalytic cycle. Although the overall reaction is quite exergonic, we show that during the reduction of molecular oxygen in cNOR, two of the reduction steps are endergonic with high barriers for proton uptake, which is in contrast to oxygen reduction in CcO, where all reduction steps are exergonic. This difference between the two enzymes is suggested to be important for their differing capabilities for energy conservation. An additional result from this study is that at least three of the four reduction steps are initiated by proton transfer to the active site, which is in contrast to CcO, where electrons always arrive before the protons to the active site. The roles of the non-heme metal ion and the redox-active tyrosine in the active site are also discussed.

Functional heterogeneity of Fo·F1H+-ATPase/synthase in coupled Paracoccus denitrificans plasma membranes.

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.

A simulation-guided fluorescence correlation spectroscopy tool to investigate the protonation dynamics of cytochrome c oxidase.

Fluorescence correlation spectroscopy (FCS) is a single molecule based technique to temporally resolve rate-dependent processes by correlating the fluorescence fluctuations of individual molecules traversing through a confocal volume. In addition, chemical processes like protonation or intersystem crossing can be monitored in the sub-microsecond range. FCS thereby provides an excellent tool for investigations of protonation dynamics in proton pumps like cytochrome c oxidase (CcO). To achieve this, the pH-dependent fluorescent dye fluorescein was attached as a protonation probe to the CcO surface via site-specific labeling of single reactive cysteines that are located close to the entry point of a proton input channel (K-pathway). The analysis of protonation dynamics is complicated by overlapping triplet and protonation rates of the fluorophore. A Monte Carlo simulation based algorithm was developed to facilitate discrimination of these temporally overlapping processes thus allowing for improved protonation reaction rate determination. Using this simulation-guided approach we determined precise local proton association and dissociation rates and provide information about protein surface effects, such as proton collecting antennae, on the transport properties of proton transfer channels.

Biomimetic Membranes for Multi-Redox Center Proteins.

His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs). An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA) terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM). MRPs, such as the cytochrome c oxidase (CcO) from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs) from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced resonance Raman spectroscopy (SERRS), were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs). The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM) and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

Homeostatic control of nitric oxide (NO) at nanomolar concentrations in denitrifying bacteria - modelling and experimental determination of NO reductase kinetics in vivo in Paracoccus denitrificans.

Homeostatic control of nitric oxide (NO) at nanomolar concentrations appears common among denitrifying bacteria, often ascribed to synchronized expression of nitrite and nitric oxide reductase (Nir and Nor). We questioned whether this is sufficient: using the reported substrate affinities for cytochrome cd1 nitrite reductase (cNor), our model of batch cultures of Paracoccus denitrificans predicted NO concentrations orders of magnitude higher than measured. We rejected a hypothesis that the homeostatic control is due to a negative feedback by NO on the activity of NirS because the inclusion of such feedback resulted in too slow anaerobic growth and N2 production. We proceeded by determining the kinetic parameters for cNor in vivo by a carefully designed experiment, allowing the estimation of NO concentration at the cell surface while anoxic cultures depleted low headspace doses of NO. With the new parameters for cNor kinetics in vivo {v = vmax /[1 + K2 /(NO) + K1 × K2 /(NO)(2) ]; vmax = 3.56 fmol NO cell(-1) h(-1) , K1 < 1 nM, and K2 = 34 nM}, the model predicted NO concentrations close to that measured. Thus, enzyme kinetics alone can explain the observed NO homeostasis. Determinations of enzyme kinetic parameters in vivo are not trivial but evidently required to understand and model NO kinetics in denitrifying organisms in soils and aquatic environments.

NMR structures of α-proteobacterial ATPase-regulating ζ-subunits.

NMR structures of ζ-subunits, which are recently discovered α-proteobacterial F1F0-ATPase-regulatory proteins representing a Pfam protein family of 246 sequences from 219 species (PF07345), exhibit a four-helix bundle, which is different from all other known F1F0-ATPase inhibitors. Chemical shift mapping reveals a conserved ADP/ATP binding site in ζ-subunit, which mediates long-range conformational changes related to function, as revealed by the structure of the Paracoccus denitrificans ζ-subunit in complex with ADP. These structural data suggest a new mechanism of F1F0-ATPase regulation in α-proteobacteria.

The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site.

The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it is separated by 10.1Å. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pKa value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the ß-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence.

Oxidative damage in MauG: implications for the control of high-valent iron species and radical propagation pathways.

The di-heme enzyme MauG catalyzes the oxidative biosynthesis of a tryptophan tryptophylquinone cofactor on a precursor of the enzyme methylamine dehydrogenase (preMADH). Reaction of H2O2 with the diferric form of MauG, or reaction of O2 with diferrous MauG, forms the catalytic intermediate known as bis-Fe(IV), which acts as the key oxidant during turnover. The site of substrate oxidation is more than 40 Å from the high-spin heme iron where H2O2 initially reacts, and catalysis relies on radical hopping through an interfacial residue, Trp199 of MauG. In the absence of preMADH, the bis-Fe(IV) intermediate is remarkably stable, but repeated exposure to H2O2 results in suicide inactivation. Using mass spectrometry, we show that this process involves the oxidation of three Met residues (108, 114, and 116) near the high-spin heme through ancillary electron transfer pathways engaged in the absence of substrate. The mutation of a conserved Pro107 in the distal pocket of the high-spin heme results in a dramatic increase in the level of oxidation of these Met residues. These results illustrate structural mechanisms by which MauG controls reaction with its high-valent heme cofactor and limits uncontrolled oxidation of protein residues and loss of catalytic activity. The conservation of Met residues near the high-spin heme among MauG homologues from different organisms suggests that eventual deactivation of MauG may function in a biological context. That is, methionine oxidation may represent a protective mechanism that prevents the generation of reactive oxygen species by MauG in the absence of preMADH.

Simultaneous quantification of energetically important metabolites in various cell types by CZE.

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.

Keeping PASE with WEFT: SHWEFT-PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules.

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from (1)H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T1 and T2. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High-spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one-dimensional (1D) pulse sequences combine shaped pulses with super-water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super-water elimination Fourier transform-paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple (1)H 1D, 1D NOE, and two-dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co(2+)-amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high-spin metBjFixLH(151-256), the heme sensing domain of Bradyrhizobium japonicum FixL.

Unexpected weak magnetic exchange coupling between haem and non-haem iron in the catalytic site of nitric oxide reductase (NorBC) from Paracoccus denitrificans1.

Bacterial NOR (nitric oxide reductase) is a major source of the powerful greenhouse gas N2O. NorBC from Paracoccus denitrificans is a heterodimeric multi-haem transmembrane complex. The active site, in NorB, comprises high-spin haem b3 in close proximity with non-haem iron, FeB. In oxidized NorBC, the active site is EPR-silent owing to exchange coupling between FeIII haem b3 and FeBIII (both S=5/2). On the basis of resonance Raman studies [Moënne-Loccoz, Richter, Huang, Wasser, Ghiladi, Karlin and de Vries (2000) J. Am. Chem. Soc. 122, 9344-9345], it has been assumed that the coupling is mediated by an oxo-bridge and subsequent studies have been interpreted on the basis of this model. In the present study we report a VFVT (variable-field variable-temperature) MCD (magnetic circular dichroism) study that determines an isotropic value of J=-1.7 cm-1 for the coupling. This is two orders of magnitude smaller than that encountered for oxo-bridged diferric systems, thus ruling out this configuration. Instead, it is proposed that weak coupling is mediated by a conserved glutamate residue.

Characterization of electron tunneling and hole hopping reactions between different forms of MauG and methylamine dehydrogenase within a natural protein complex.

Respiration, photosynthesis, and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate that is sufficient to support the biological activity. A multistep hole hopping mechanism could, in principle, enhance the efficiency of long-range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally determined ET distances matched the distances determined from the crystal structure that would be expected for single-step tunneling and multistep hopping. Experimentally determined relative values of electronic coupling (H(AB)) for the two reactions correlated well with the relative H(AB) values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also 10-fold greater than that of the single-step tunneling reaction despite a smaller overall driving force for the hopping-mediated ET reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multistep hopping.