RESUMEN
Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.
Asunto(s)
Grupo Citocromo c/metabolismo , Citocromos/metabolismo , Electrones , Hemo/metabolismo , Histidina/metabolismo , Metionina/metabolismo , Shewanella/metabolismo , Grupo Citocromo c/química , Citocromos/química , Transporte de Electrón , Hemo/química , Histidina/química , Metionina/química , Simulación de Dinámica Molecular , Nanocables , Oxidación-ReducciónRESUMEN
A growing number of bacterial species are known to move electrons across their cell envelopes. Naturally this occurs in support of energy conservation and carbon-fixation. For biotechnology it allows electron exchange between bacteria and electrodes in microbial fuel cells and during microbial electrosynthesis. In this context Rhodopseudomonas palustris TIE-1 is of much interest. These bacteria respond to light by taking electrons from their external environment, including electrodes, to drive CO2-fixation. The PioA cytochrome, that spans the bacterial outer membrane, is essential for this electron transfer and yet little is known about its structure and electron transfer properties. Here we reveal the ten c-type hemes of PioA are redox active across the window +250 to -400 mV versus Standard Hydrogen Electrode and that the hemes with most positive reduction potentials have His/Met and His/H2O ligation. These chemical and redox properties distinguish PioA from the more widely studied family of MtrA outer membrane decaheme cytochromes with ten His/His ligated hemes. We predict a structure for PioA in which the hemes form a chain spanning the longest dimension of the protein, from Heme 1 to Heme 10. Hemes 2, 3 and 7 are identified as those most likely to have His/Met and/or His/H2O ligation. Sequence analysis suggests His/Met ligation of Heme 2 and/or 7 is a defining feature of decaheme PioA homologs from over 30 different bacterial genera. His/Met ligation of Heme 3 appears to be less common and primarily associated with PioA homologs from purple non-sulphur bacteria belonging to the alphaproteobacteria class.
Asunto(s)
Citocromos/química , Citocromos/metabolismo , Hemo/química , Rhodopseudomonas/fisiología , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Técnicas Electroquímicas , Transporte de Electrón , Modelos Moleculares , Fotosíntesis , Conformación ProteicaRESUMEN
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 × 106 s-1 was resolved for Heme IV â 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 × 106 s-1 was defined for Heme I â 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 â¼6 (11) Å. The results are significant in demonstrating the opportunities for pump-probe spectroscopies to resolve interheme electron transfer in Ru-labeled multiheme cytochromes.
Asunto(s)
Complejos de Coordinación/metabolismo , Citocromos/metabolismo , Luz , Complejos de Coordinación/química , Citocromos/química , Transporte de Electrón , Simulación de Dinámica MolecularRESUMEN
Multiheme cytochromes possess closely packed redox-active hemes arranged as chains spanning the tertiary structure. Here we describe five variants of a representative multiheme cytochrome engineered as biohybrid phototransducers for converting light into electricity. Each variant possesses a single Cys sulfhydryl group near a terminus of the heme chain, and this was efficiently labelled with a RuII (2,2'-bipyridine)3 photosensitiser. When irradiated in the presence of a sacrificial electron donor (SED) the proteins exhibited different types of behaviour. Certain proteins were rapidly and fully reduced. Other proteins were rapidly semi-reduced but resisted complete photoreduction. These findings reveal that photosensitised multiheme cytochromes can be engineered to act as resistors, with intrinsic regulation of light-driven electron accumulation, and also as molecular wires with essentially unhindered photoreduction. It is proposed that the observed behaviour arises from interplay between the site of electron injection and the distribution of heme reduction potentials along the heme chain.
Asunto(s)
Grupo Citocromo c/química , Transporte de Electrón , Hemo/química , Fototransducción , Shewanella/metabolismo , Grupo Citocromo c/genética , Electrones , Cinética , Fármacos Fotosensibilizantes , Shewanella/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Hemo/química , Hierro/química , Oxidorreductasas/química , Paracoccus denitrificans/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Hemo/metabolismo , Hierro/metabolismo , Cinética , Fenómenos Magnéticos , Oxidación-Reducción , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Paracoccus denitrificans/enzimología , TermodinámicaRESUMEN
Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. This redox-driven proton pump catalyzes the four-electron reduction of molecular oxygen to water, one of the most fundamental processes in biology. Elucidation of the intermediate structures in the catalytic cycle is crucial for understanding both the mechanism of oxygen reduction and its coupling to proton pumping. Using CcO from Paracoccus denitrificans, we demonstrate that the artificial F state, classically generated by reaction with an excess of hydrogen peroxide, can be converted into a new P state (in contradiction to the conventional direction of the catalytic cycle) by addition of ammonia at pH 9. We suggest that ammonia coordinates directly to Cu(B) in the binuclear active center in this P state and discuss the chemical structures of both oxoferryl intermediates F and P. Our results are compatible with a superoxide bound to Cu(B) in the F state.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Paracoccus denitrificans/enzimología , Amoníaco/metabolismo , Biocatálisis , Espectroscopía de Resonancia por Spin del Electrón , Complejo IV de Transporte de Electrones/química , Concentración de Iones de Hidrógeno , Oxígeno/metabolismoRESUMEN
Multiheme cytochromes (MHCs) are the building blocks of highly conductive micrometre-long supramolecular wires found in so-called electrical bacteria. Recent studies have revealed that these proteins possess a long supramolecular array of closely packed heme cofactors along the main molecular axis alternating between perpendicular and stacking configurations (TST = T-shaped, stacked, T-shaped). While TST arrays have been identified as the likely electron conduit, the mechanisms of outstanding long-range charge transport observed in these structures remain unknown. Here we study charge transport on individual small tetraheme cytochromes (STCs) containing a single TST heme array. Individual STCs are trapped in a controllable nanoscale tunnelling gap. By modulating the tunnelling gap separation, we are able to selectively probe four different electron pathways involving 1, 2, 3 and 4 heme cofactors, respectively, leading to the determination of the electron tunnelling decay constant along the TST heme motif. Conductance calculations of selected single-STC junctions are in excellent agreement with experiments and suggest a mechanism of electron tunnelling with shallow length decay constant through an individual STC. These results demonstrate that an individual TST motif supporting electron tunnelling might contribute to a tunnelling-assisted charge transport diffusion mechanism in larger TST associations.
RESUMEN
Shewanella oneidensis exchanges electrons between cellular metabolism and external redox partners in a process that attracts much attention for production of green electricity (microbial fuel cells) and chemicals (microbial electrosynthesis). A critical component of this pathway is the outer membrane spanning MTR complex, a biomolecular wire formed of the MtrA, MtrB, and MtrC proteins. MtrA and MtrC are decaheme cytochromes that form a chain of close-packed hemes to define an electron transfer pathway of 185 Å. MtrA is wrapped inside MtrB for solubility across the outer membrane lipid bilayer; MtrC sits outside the cell for electron exchange with external redox partners. Here, we demonstrate tight and spontaneous in vitro association of MtrAB with separately purified MtrC. The resulting complex is comparable with the MTR complex naturally assembled by Shewanella in terms of both its structure and rates of electron transfer across a lipid bilayer. Our findings reveal the potential for building bespoke electron conduits where MtrAB combines with chemically modified MtrC, in this case, labeled with a Ru-dye that enables light-triggered electron injection into the MtrC heme chain.
RESUMEN
Microbial nanowires are fascinating biological structures that allow bacteria to transport electrons over micrometers for reduction of extracellular substrates. It was recently established that the nanowires of both Shewanella and Geobacter are made of multi-heme proteins; but, while Shewanella employs the 20-heme protein complex MtrCAB, Geobacter uses a redox polymer made of the hexa-heme protein OmcS, begging the question as to which protein architecture is more efficient in terms of long-range electron transfer. Using a multiscale computational approach we find that OmcS supports electron flows about an order of magnitude higher than MtrCAB due to larger heme-heme electronic couplings and better insulation of hemes from the solvent. We show that heme side chains are an essential structural element in both protein complexes, accelerating rate-limiting electron tunnelling steps up to 1000-fold. Our results imply that the alternating stacked/T-shaped heme arrangement present in both protein complexes may be an evolutionarily convergent design principle permitting efficient electron transfer over very long distances.
Asunto(s)
Proteínas Bacterianas/química , Hemo/química , Hemoproteínas/química , Transporte de Electrón , Geobacter/química , Nanocables/química , Oxidación-Reducción , Conformación Proteica , Shewanella/química , Solventes/química , Relación Estructura-ActividadRESUMEN
Multi-heme cytochromes (MHCs) are fascinating proteins used by bacterial organisms to shuttle electrons within, between, and out of their cells. When placed in solid-state electronic junctions, MHCs support temperature-independent currents over several nanometers that are 3 orders of magnitude higher compared to other redox proteins of similar size. To gain molecular-level insight into their astonishingly high conductivities, we combine experimental photoemission spectroscopy with DFT+Σ current-voltage calculations on a representative Gold-MHC-Gold junction. We find that conduction across the dry, 3 nm long protein occurs via off-resonant coherent tunneling, mediated by a large number of protein valence-band orbitals that are strongly delocalized over heme and protein residues. This picture is profoundly different from the electron hopping mechanism induced electrochemically or photochemically under aqueous conditions. Our results imply that the current output in solid-state junctions can be even further increased in resonance, for example, by applying a gate voltage, thus allowing a quantum jump for next-generation bionanoelectronic devices.
Asunto(s)
Hemoproteínas/química , Citocromos/química , Teoría Funcional de la Densidad , Conductividad Eléctrica , Técnicas Electroquímicas , Transporte de Electrón , Oro/química , Hemo/química , Modelos Moleculares , Oxidación-Reducción , Procesos Fotoquímicos , Conformación Proteica , AguaRESUMEN
Multi-heme cytochrome c (Cytc) proteins are key for transferring electrons out of cells, to enable intracellular oxidation to proceed in the absence of O2. In these proteins most of the hemes are arranged in a linear array suggesting a facile path for electronic conduction. To test this, we studied solvent-free electron transport across two multi-heme Cytc-type proteins: MtrF (deca-heme Cytc) and STC (tetra-heme Cytc). Transport is measured across monolayers of these proteins in a solid state configuration between Au electrodes. Both proteins showed 1000× higher conductance than single heme, or heme-free proteins, but similar conductance to monolayers of conjugated organics. Conductance is found to be temperature-independent (320-80 K), suggesting tunneling as the transport mechanism. This mechanism is consistent with I-V curves modelling, results of which could be interpreted by having protein-electrode coupling as rate limiting, rather than transport within the proteins.
RESUMEN
Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions can proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering, and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Transporte de Electrón/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Oxidación-Reducción , Shewanella/enzimologíaRESUMEN
Prokaryotic metal-ion receptor proteins, or solute-binding proteins, facilitate the acquisition of metal ions from the extracellular environment. Pneumococcal surface antigen A (PsaA) is the primary Mn(2+)-recruiting protein of the human pathogen Streptococcus pneumoniae and is essential for its in vivo colonization and virulence. The recently reported high-resolution structures of metal-free and metal-bound PsaA have provided the first insights into the mechanism of PsaA-facilitated metal binding. However, the conformational dynamics of metal-free PsaA in solution remain unknown. Here, we use continuous wave electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations to study the relative flexibility of the structural domains in metal-free PsaA and its distribution of conformations in solution. The results show that the crystal structure of metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also samples structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is both larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomic-resolution insight into the conformational dynamics of PsaA prior to metal binding and lays the groundwork for future EPR and MD based studies of PsaA in solution.
Asunto(s)
Adhesinas Bacterianas/química , Lipoproteínas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Lipoproteínas/genética , Lipoproteínas/metabolismo , Manganeso/química , Metales/química , Simulación de Dinámica Molecular , Mutagénesis , Estructura Terciaria de Proteína , Marcadores de SpinRESUMEN
Understanding the process that underlies multidrug recognition and efflux by P-glycoprotein (ABCB1) remains a key biological challenge. Structural data have recently become available for the murine and Caenorhabditis elegans homologues of ABCB1; however all structures were obtained in the absence of nucleotide. A feature of these structures was the presence of a central cavity that is inaccessible from the extracellular face of the protein. To determine the conformational dynamics of this region several residues in transmembrane helices TM6 (331, 343 and 354) and TM12 (980) were mutated to cysteine. Based upon structural predictions, these residues are proposed to line, or reside proximal to, the central cavity. The mutant isoforms were labelled with a paramagnetic probe enabling the application of EPR spectroscopic methods. Power saturation EPR spectra were recorded in the presence of hydrophobic (O2 ) or hydrophilic (NiEDDA) quenching agents to study the local environment of each residue. ABCB1 was trapped in both its nucleotide-bound and post-hydrolytic conformations and EPR spectra were again recorded in the presence and absence of quenching agents. The EPR line shapes provide information on the movements of these residues within TM6 and TM12 during ATP hydrolysis. Rationalization of the data with molecular dynamic simulations indicates that the cavity is converted to a configuration open to the aqueous phase following nucleotide binding, thereby suggesting alternating access to the cavity on opposite sides of the membrane during translocation.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Animales , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Insectos , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO. than the parent strain led to the proposal that NrfA might also participate in NO. detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO. reductase activity. NO. reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO. reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO. reduction by NrfA is described by a K(m) of the order of 300 microm. The predicted turnover number of approximately 840 NO. s(-1) is much higher than that of the dedicated respiratory NO. reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO. detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO. encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO. levels such that any NO. reaching the cytoplasm is efficiently removed by flavorubredoxin (K(m) approximately 0.4 microm).
Asunto(s)
Grupo Citocromo c/metabolismo , Escherichia coli/enzimología , Óxido Nítrico/metabolismo , Oxidorreductasas/metabolismo , Proteínas Periplasmáticas/metabolismo , Anaerobiosis/fisiología , Grupo Citocromo c/genética , Escherichia coli/crecimiento & desarrollo , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/genética , Humanos , Hidroxilamina/metabolismo , Mutación , Nitritos/metabolismo , Oxidorreductasas/genética , Proteínas Periplasmáticas/genéticaRESUMEN
The pentaheme cytochrome c nitrite reductase (NrfA) of Escherichia coli is responsible for nitrite reduction during anaerobic respiration when nitrate is scarce. The NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a glutamine (Q263) positioned 8 A from the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion. Mutation of this glutamine to the more usual calcium ligand, glutamate, results in an increase in the K m for nitrite by around 10-fold, while V max is unaltered. Protein film voltammetry showed that lower potentials were required to detect activity from NrfA Q263E when compared with native enzyme, consistent with the introduction of a negative charge into the vicinity of the active site heme. EPR and MCD spectroscopic studies revealed the high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme. Comparison of the X-ray crystal structures of the as-prepared, oxidized native and mutant enzymes showed an increased bond distance between the active site heme Fe(III) iron and the distal ligand in the latter as well as changes to the structure and mobility of the active site water molecule network. These results suggest that an important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand.
Asunto(s)
Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Glutamina/química , Sustitución de Aminoácidos , Sitios de Unión , Catálisis , Dicroismo Circular , Cristalización , Cristalografía por Rayos X , Grupo Citocromo c/genética , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/enzimología , Ácido Glutámico/química , Glutamina/fisiología , Modelos MolecularesRESUMEN
Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.
Asunto(s)
Citocromos/química , Citocromos/metabolismo , Hemo/análogos & derivados , Nitrito Reductasas/química , Nitrito Reductasas/metabolismo , Oxígeno/química , Paracoccus pantotrophus/enzimología , Dicroismo Circular , Grupo Citocromo c , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Hemo/química , Ligandos , Modelos Químicos , Nitritos/química , Pseudomonas stutzeri/enzimología , Espectrofotometría , TemperaturaRESUMEN
Hemoproteins have been recognized for nearly a century and are ubiquitous components of cellular organisms. Despite our familiarity with these proteins, defining the functional role of a given heme can still present considerable challenges. In this situation, magnetic circular dichroism (MCD) is a technique of choice because it has the capacity to define heme oxidation, spin, and ligation states in solution and at ambient temperature. Unfortunately, the resolving power of MCD rarely has been brought to bare on the intermediate redox states accessible to multiheme proteins. This is due in large part to the time-consuming procedure of magnetic field cycling required each time a sample is introduced into the magnet and the risk that control over, and knowledge of, the potential will be lost between sample preparation and spectral acquisition. Here we present a solution to this problem in the form of MCD-compatible optically transparent thin-layer electrochemistry (MOTTLE). MOTTLE defines redox behavior for cytochrome c in good agreement with the literature. In addition, MOTTLE reproduces the redox-driven transformation of heme ligand sets reported for cytochrome bd. Thus, MOTTLE provides a robust analytical tool for the dissection of heme properties with resolution across the electrochemical potential domain.