Regulation and Characterization of Mutants of fixABCX in Rhizobium leguminosarum.

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5'-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin.

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans.

Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd1 nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd1NiR, presumably because cd1NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd1NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd1NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd1NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.

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

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 → 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 → Heme II electron transfer. These rates are an order of magnitude faster than previously computed values for unlabeled STC. The Heme III/IV and I/II pairs exemplify the T-shaped heme packing arrangement, prevalent in multiheme cytochromes, whereby the adjacent porphyrin rings lie at 90° with edge-edge (Fe-Fe) distances of ∼6 (11) Å. The results are significant in demonstrating the opportunities for pump-probe spectroscopies to resolve interheme electron transfer in Ru-labeled multiheme cytochromes.

Photosensitised Multiheme Cytochromes as Light-Driven Molecular Wires and Resistors.

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.

Tuning the modular Paracoccus denitrificans respirome to adapt from aerobic respiration to anaerobic denitrification.

Bacterial denitrification is a respiratory process that is a major source and sink of the potent greenhouse gas nitrous oxide. Many denitrifying bacteria can adjust to life in both oxic and anoxic environments through differential expression of their respiromes in response to environmental signals such as oxygen, nitrate and nitric oxide. We used steady-state oxic and anoxic chemostat cultures to demonstrate that the switch from aerobic to anaerobic metabolism is brought about by changes in the levels of expression of relatively few genes, but this is sufficient to adjust the configuration of the respirome to allow the organism to efficiently respire nitrate without the significant release of intermediates, such as nitrous oxide. The regulation of the denitrification respirome in strains deficient in the transcription factors FnrP, Nnr and NarR was explored and revealed that these have both inducer and repressor activities, possibly due to competitive binding at similar DNA binding sites. This may contribute to the fine tuning of expression of the denitrification respirome and so adds to the understanding of the regulation of nitrous oxide emission by denitrifying bacteria in response to different environmental signals.

Analysis of multiple haloarchaeal genomes suggests that the quinone-dependent respiratory nitric oxide reductase is an important source of nitrous oxide in hypersaline environments.

Microorganisms, including Bacteria and Archaea, play a key role in denitrification, which is the major mechanism by which fixed nitrogen returns to the atmosphere from soil and water. While the enzymology of denitrification is well understood in Bacteria, the details of the last two reactions in this pathway, which catalyse the reduction of nitric oxide (NO) via nitrous oxide (N2 O) to nitrogen (N2 ), are little studied in Archaea, and hardly at all in haloarchaea. This work describes an extensive interspecies analysis of both complete and draft haloarchaeal genomes aimed at identifying the genes that encode respiratory nitric oxide reductases (Nors). The study revealed that the only nor gene found in haloarchaea is one that encodes a single subunit quinone dependent Nor homologous to the qNor found in bacteria. This surprising discovery is considered in terms of our emerging understanding of haloarchaeal bioenergetics and NO management.

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids.

UNLABELLED: Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2 Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [(13)C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-ß-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids. IMPORTANCE: Biological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N2 However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-ß-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules.

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.

Structure of a bacterial cell surface decaheme electron conduit.

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split ß-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

Electron transfer to the active site of the bacterial nitric oxide reductase is controlled by ligand binding to heme b3.

The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b3 and non heme iron (Fe(B)). These metal centres are shown to be at isopotential with midpoint reduction potentials of E(m) ≈ +80 mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b, are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b3 is raised to a region similar to that of heme c and heme b. This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding.

Enzymology and ecology of the nitrogen cycle.

The nitrogen cycle describes the processes through which nitrogen is converted between its various chemical forms. These transformations involve both biological and abiotic redox processes. The principal processes involved in the nitrogen cycle are nitrogen fixation, nitrification, nitrate assimilation, respiratory reduction of nitrate to ammonia, anaerobic ammonia oxidation (anammox) and denitrification. All of these are carried out by micro-organisms, including bacteria, archaea and some specialized fungi. In the present article, we provide a brief introduction to both the biochemical and ecological aspects of these processes and consider how human activity over the last 100 years has changed the historic balance of the global nitrogen cycle.

Electron transfer and half-reactivity in nitrogenase.

Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum-iron protein (MoFe protein) and a protein containing an [Fe4-S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

Mutagenesis of tyrosine residues within helix VII in subunit I of the cytochrome cbb3 oxidase from Rhodobacter capsulatus.

The cbb (3)-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. The cbb (3) oxidases are missing an active-site tyrosine residue that is absolutely conserved in all A and B-type heme-copper oxidases. This tyrosine is known to play a critical role in the catalytic mechanisms of A and B-type oxidases. The absence of this tyrosine in the cbb (3) oxidases raises the possibility that the cbb (3) oxidases utilize a different catalytic mechanism from that of the other members of the superfamily, or have this conserved residue in different helices. Recently sequence comparisons indicate that, a tyrosine residues that might be analogous to the active-site tyrosine in other oxidases are present in the cbb (3) oxidases but these tyrosines originates from a different transmembrane helix within the protein. In this research, three conserved tyrosine residues, Y294, Y308 and Y318, in helix VII were substituted for phenylalanine. Y318F mutant in the Rhodobacter capsulatus oxidase resulted in a fully assembled enzyme with nativelike structure and activity, but Y294F mutant is not assembled and have a catalytic activity. On the other hand, Y308F mutant is fully assembled enzyme with nativelike structure, but lacking catalytic activity. This result indicates that Y308 should be crucial in catalytic activity of the cbb (3) oxidase of R. capsulatus. These findings support the assumption that all of the heme-copper oxidases utilize the same catalytic mechanism and provide a residue originates from different places within the primary sequence for different members of the same superfamily.

Mechanistic insight into the nitrosylation of the [4Fe-4S] cluster of WhiB-like proteins.

The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.

The electron transfer flavoprotein: ubiquinone oxidoreductases.

Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).

Molecular dissection of bacterial acrylate catabolism--unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production.

A bacterium in the genus Halomonas that grew on dimethylsulfoniopropionate (DMSP) or acrylate as sole carbon sources and that liberated the climate-changing gas dimethyl sulfide in media containing DMSP was obtained from the phylloplane of the macroalga Ulva. We identified a cluster that contains genes specifically involved in DMSP catabolism (dddD, dddT) or in degrading acrylate (acuN, acuK) or that are required to break down both substrates (dddC, dddA). Using NMR and HPLC analyses to trace 13C- or 14C-labelled acrylate and DMSP in strains of Escherichia coli with various combinations of cloned ddd and/or acu genes, we deduced that DMSP is imported by the BCCT-type transporter DddT, then converted by DddD to 3-OH-propionate (3HP), liberating dimethyl sulfide in the process. As DddD is a predicted acyl CoA transferase, there may be an earlier, unidentified catabolite of DMSP. Acrylate is also converted to 3HP, via a CoA transferase (AcuN) and a hydratase (AcuK). The 3HP is predicted to be catabolized by an alcohol dehydrogenase, DddA, to malonate semialdehyde, thence by an aldehyde dehydrogenase, DddC, to acyl CoA plus CO2. The regulation of the ddd and acu genes is unusual, as a catabolite, 3HP, was a co-inducer of their transcription. This first description of genes involved in acrylate catabolism in any organism shows that the relationship between the catabolic pathways of acrylate and DMSP differs from that which had been suggested in other bacteria.

Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase.

The c-type nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans is an integral membrane protein that catalyzes NO reduction; 2NO+2e(-)+2H(+)-->N(2)O+H(2)O. It is also capable of catalyzing the reduction of oxygen to water, albeit more slowly than NO reduction. cNORs are divergent members of the heme-copper oxidase superfamily (HCuOs) which reduce NO, do not pump protons, and the reaction they catalyse is non-electrogenic. All known cNORs have been shown to have five conserved glutamates (E) in the catalytic subunit, by P. denitrificans numbering, the E122, E125, E198, E202 and E267. The E122 and E125 are presumed to face the periplasm and the E198, E202 and E267 are located in the interior of the membrane, close to the catalytic site. We recently showed that the E122 and E125 define the entry point of the proton pathway leading from the periplasm into the active site [U. Flock, F.H. Thorndycroft, A.D. Matorin, D.J. Richardson, N.J. Watmough, P. Adelroth, J. Biol. Chem. 283 (2008) 3839-3845]. Here we present results from the reaction between fully reduced NOR and oxygen on the alanine variants of the E198, E202 and E267. The initial binding of O(2) to the active site was unaffected by these mutations. In contrast, proton uptake to the bound O(2) was significantly inhibited in both the E198A and E267A variants, whilst the E202A NOR behaved essentially as wildtype. We propose that the E198 and E267 are involved in terminating the proton pathway in the region close to the active site in NOR.

The bacterial respiratory nitric oxide reductase.

The two-subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best-characterized example of the bacterial respiratory nitric oxide reductases. These are members of the super-family of haem-copper oxidases and are characterized by the elemental composition of their active site, which contains non-haem iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalysed. The reaction requires the presence of two substrate molecules at the active site along with the controlled input of two electrons and two protons from the same side of the membrane. In the present paper, we consider progress towards understanding the pathways of electron and proton transfer in NOR and how this information can be integrated with evidence for the likely modes of substrate binding at the active site to propose a revised and experimentally testable reaction mechanism.

Electrochemical evidence for multiple peroxidatic heme states of the diheme cytochrome c peroxidase of Pseudomonas aeruginosa.

The enzyme cytochrome c peroxidase from Pseudomonas aeruginosa and its catalytic mechanism were investigated using protein film voltammetry. Monolayers of the diheme bacterial enzyme were immobilized on both pyrolytic graphite edge and alkanethiol-modified Au electrodes. The redox couple associated with the low potential heme could be detected on both electrode surfaces at a reduction potential of -234 mV vs SHE. The midpoint potential displays a distinct pH dependence at acidic pH values, indicative of proton-coupled electron transfer. The nonturnover signal of the LP heme can be transformed into sigmoidal waves upon the addition of substrate. The midpoint potentials of the turnover signals were used to calculate Michaelis-Menten kinetics with a K(m) = 25 microM. Catalysis was inhibited with addition of cyanide (K(i) = 50 microM). These kinetic parameters are in good agreement with previously reported solution-based studies, indicating that the activity of the enzyme is unaffected by the immobilization on the electrode surface. The reduction potential of the catalytic wave clearly shows that the rate-limiting species during electrocatalysis differs from those previously reported for peroxidases, indicating that PFV may be used in the future to distinguish the requirement for reductive activation in bacterial cytochrome c peroxidases.