His/Met heme ligation in the PioA outer membrane cytochrome enabling light-driven extracellular electron transfer by Rhodopseudomonas palustris TIE-1

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.


Introduction
Iron is an abundant component of marine, freshwater and subsurface habitats where the Fe(III)/(II) reduction potential has values between approx. −300 and +800 mV versus Standard Hydrogen Electrode (SHE) [1]. This property, arising from the different iron coordination spheres of its mineral and complexed forms, benefits the numerous microorganisms able to harness this couple for energy metabolism [1][2][3][4][5]. Examples are found in the reduction of Fe(III) that terminates anaerobic respiratory electron transfer chains in dissimilatory iron reducers, and in the oxidation of Fe(II) providing electrons for CO 2 -fixation by lithoautotrophic iron oxidisers. To use the Fe(III)/(II) transition in these ways, the corresponding organisms have evolved to manage the insolubility of Fe(III) at circum-neutral pH [6]. Lithoautotrophs avoid the formation of intracellular Fe(III) precipitates by catalysing Fe(II) oxidation on the cellular surface. Mineral forms of Fe(III) are reduced for anaerobic respiration when dissimilatory iron reducers move electrons to the cell surface. Both strategies rely on electron exchange across the cell envelope to couple the redox activities of intracellular enzymes with extracellular iron.
For several species of dissimilatory iron reducing Gramnegative bacteria, genetic and biochemical studies have demonstrated a defining role for porin:cytochrome complexes in electron transfer across the outer membrane lipid bilayer [4,[7][8][9][10]. The first molecular structure for such a complex was very recently resolved [11] and this revealed an insulated biomolecular wire for the MtrA:MtrB complex from Shewanella baltica OS185 (figure 1(A) left). At the core of that complex is a chain of ten close-packed c-hemes. This heme chain spans the lipid bilayer allowing complementary Fe(III) ↔ Fe(II) transitions of neighbouring sites to transfer electrons from the periplasm to the cell exterior. All ten hemes have His/His ligation provided by MtrA residues and they are covalently linked to CxxCH c-heme binding amino acid motifs in the same protein. MtrB is a β-barrel porin that forms a sleeve round MtrA. MtrB confers solubility in the hydrophobic membrane interior and precludes hemes in the centre of the MtrA wire from undergoing interprotein electron transfer.
Homologs of the Shewanella mtrA-mtrB genes are also found in Rhodopseudomonas palustris TIE-1 [14,15] and Rhodomicrobium vannielii [4] where they are termed pioA-pioB. The pio gene products allow phototrophic Fe(II)oxidation (figure 1(A) right), giving insight into an ancient form of photosynthesis, and supporting light-driven CO 2fixation coupled to cathodic electron influx [12,[16][17][18]]. An outer membrane porin of ∼800 amino acids similar to MtrB is predicted from pioB. The predicted gene product from pioA is larger than MtrA, ∼540 and 300 residues respectively ( figure 1(B)). However post-secretory proteolysis of the PioA N-terminus results in a mature functional cytochrome of similar size to MtrA [14,19]. Here we present the first detailed description of the structure-function relationship for PioA. Spectroscopic, redox and structural properties of PioA are described and found to differ from the corresponding properties of the well-studied MtrA proteins [7,11]. The results provide molecular insight into differences between homologous proteins that have evolved to move electrons in to or out from bacteria, PioA and MtrA respectively.

Purification of PioA as a monomeric 40 kDa decaheme cytochrome
Heterologous production of several different multiheme cytochromes in MR-1 has been achieved with pBAD202 plasmids allowing expression from an L-arabinose inducible promoter [20][21][22]. As R. palustris TIE-1 is a slow growing organism that reaches very low biomass compared to MR-1 [14,19,23], we investigated the latter organism for heterologous production of PioA. As described in Experimental Procedures, pBAD202 was engineered to carry the codon optimized R. palustris TIE-1 pioA gene with a 3 ′ extension encoding for a C-terminal Strep-II affinity tag (figures S2 and S3 (available online at stacks.iop.org/Nano/31/354002/mmedia)) to facilitate protein purification. The resulting plasmid, pBAD202_pioA was introduced into MR-1 by electroporation.  (11)) highlighting MtrA with 10 c-type hemes (red spheres with Fe black) running through the MtrB barrel by omission of beta-strands on the front face of MtrB. Schematic of S. lithotrophicus MtoAB (centre) and R. palustris PioAB (right). Large arrows indicate the direction of natural electron transfer through each complex, see text and (6) for details. For PioAB the proposed intracellular electron transfer pathway for carbon-fixation is illustrated (see [12] for details). (B) Cladogram of R. palustrus PioA homologs. Amino acid sequences and their relative lengths are indicated by grey lines aligned at the C-terminus. The locations of c-type heme binding motifs (CxxCH) are indicated as red bars and the predicted N-terminal Sec signal peptides are shown as blue octagons. Branches are labelled either with protein name or RefSeq protein accession code with the bacterial species given in parentheses. Branches corresponding to MtrA (S. oneidensis), PioA (R. palustris) and MtoA (S. lithotrophicus) are indicated with red labels. Cells were induced through the addition of 5 mM L-arabinose in the mid-exponential phase of growth and harvested approx. 6 h later. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) of the lysed cells combined with heme staining (figure 2(A)) revealed a c-type cytochrome migrating with an apparent mass of approx. 30 kDa which was not detected in equivalent analysis of noninduced cells, or MR-1 lacking the pBAD202_pioA plasmid. The identified cytochrome was confirmed as the major Strep-II tagged protein of arabinose induced cells when it was purified from the cell lysate by affinity chromatography ( figure 2(B)).
The purified protein had a red colour and the electronic absorbance, described below, was dominated by features characteristic of c-type hemes. Because all Cys residues encoded by pioA occur in the ten CxxCH heme-binding motifs, thiol(ate) content was analysed as an indicator of c-heme loading. If 10 conventional c-type hemes are present there will be no free thiol(ate) groups as all ten Cys sidechains will be present as thioether bonds to the corresponding hemes. A colourimetric assay revealed significantly less than 1 thiol(ate) per peptide, (e.g. figure S4) from which it was concluded that purified PioA contained 10 c-type hemes.
LC-MS revealed a homogeneous sample with an experimental mass of 39 966.4 (+/− 2)Da. A mass of 39 963.6 Da was predicted for the Strep-II tagged decaheme PioA with Ala243 as the N-terminal residue (figure S3) using Compute PI/Mw (https://web.expasy.org/compute_pi) and assuming each covalently attached heme would contribute 615.17 Da. This description of the mature cytochrome is in agreement with a recent study [19] that identified Ala243 as the Nterminal residue of PioA purified from R. palustris TIE-1. The extinction coefficient for oxidised PioA at 407 nm was calculated as 1440 mM −1 cm −1 using the pyridine-hemochrome method [24].
An assessment of the properties of PioA in solution was made using analytical centrifugation. Sedimentation equilibrium analysis at two PioA concentrations (0.5 and 5 µM) was performed at three centrifugation speeds (15 k, 18 k and 21 k rpm) and the absorbance profiles (figure 3(A) and figure S5) were well-described by the behaviour predicted for a single, non-interacting species with a weight average molecular mass of 45.5 ± 0.2 kDa. Comparing this value to the results of LC-MS indicates that PioA is a monomer under the experimental conditions. PioA (0.8 µM) was also analysed by sedimentation velocity analysis through repeated measurement of the absorbance during centrifugation at 40 k rpm (figure 3(B)). The absorbance profiles were fitted using the Lamm equation [25] providing a single peak in the c(s) distribution (figure 3(B)) that is characteristic of a homogenous sample containing a single species. That species is described by a sedimentation coefficient of 2.94 S and a diffusion coefficient of 6.48 × 10 −7 cm 2 s −1 from which a molecular weight of 43.2 kDa is calculated using the Svedberg equation as described in Experimental Procedures. Thus, sedimentation velocity analysis is also indicative of monomeric PioA. From the measured sedimentation coefficient and molecular weight, an f /f 0 ratio of 1.344 was calculated. This ratio provides a comparison of the PioA frictional coefficient, f, to that of a perfect sphere of equal mass, hydration and viscosity, f 0 , and is indicative of a prolate or oblate elliptical protein molecule with an elongated axis.

PioA is redox active between +250 and −400 mV versus SHE
Spectro-electrochemistry and cyclic voltammetry were used to study the redox properties of PioA at pH 7.4. For the former method, PioA was adsorbed on optically transparent mesoporous nanocrystalline SnO 2 electrodes [26]. Heme oxidation state was resolved by electronic absorbance spectroscopy after equilibrating the protein at numerous electrode potentials (figure 4(A)). When poised at +250 mV versus SHE the spectrum revealed features characteristic of low-spin c-type heme in the Fe(III) state. The Soret band had maximum intensity at 407 nm and a broad lower intensity α/β-band was present between 500 and 600 nm. Spectroscopic features indicative of reduced heme appeared after equilibrating the adsorbed protein below approx. +200 mV, namely, a Soret band with maximum intensity at 419 nm and sharp α/β-bands at 552 and 523 nm respectively. Below approx. −400 mV the spectra indicated complete reduction of the protein.
Plotting the extent of PioA reduction as a function of equilibration potential (figure 4(A)) suggests the majority of PioA hemes are reduced between approx. −100 and −300 mV and that 1 or 2 hemes are reduced between approx. +200 and −100 mV. However, confident deconvolution of these contributions is precluded because the spectral contribution of each heme is unknown. Therefore, a complementary view of PioA redox chemistry was sought from cyclic voltammetry. In this method each redox active site undergoing a singleelectron (n = 1) process makes an equal contribution to the peaks resolved for oxidation, and reduction, of the adsorbed protein. The relatively large irregular charging currents measured with optically transparent SnO 2 electrodes, e.g. [27], prevented confident analysis of the peaks describing redox transformation of adsorbed PioA. However, clear well-defined peaks (figure 4(B)) defining PioA redox activity were measured during cyclic voltammetry of the protein adsorbed on three-dimensional hierarchically-structured indium tin oxide electrodes [28,29].
The areas of the peaks for PioA oxidation (positive current) and reduction (negative current) were equal, indicating reversible redox transformation of the protein. Both features describe redox activity between ≈ +250 and −400 mV, the same range for which spectro-electrochemistry revealed conversion of PioA between the deca-Fe(III) and deca-Fe(II) states. Maximum currents were detected at approx. −180 mV and a shoulder on the low potential flank together with a smaller well-defined shoulder on the high potential flank reveal contributions from hemes having several different reduction potentials. We assessed how well the peaks are described by the sum of ten equal contributions from isolated sites undergoing reversible n = 1 processes using the protocol of Doyle et al [30]. Both peaks are well-described by our choice of model ( figure 4(B)). Reduction potentials (±15 mV) of +130, +45, The redox properties of PioA change very little between pH 6.4 and 9.4 ( figure S6). This behaviour suggests that protoncoupled electron transfer over this pH range is less prevalent in PioA than other multiheme cytochromes [31,32].

The high potential hemes of PioA have His/Met and His/H 2 O ligation
Further insight into the nature of the PioA hemes was gained from the electronic absorbance displayed by solutions of the protein prepared in different oxidation states (figure 5(A)). For these experiments we compared the spectral properties of solutions containing PioA equilibrated with air, an excess of the mild reductant ascorbate or an excess of the strong reductant dithionite.
Solutions of air-oxidised PioA displayed spectral features typical of oxidised c-type hemes and indistinguishable from those of the adsorbed protein; a Soret-band maximum at 407 nm with a broad α-/β-band between approx. 500 and 600 nm. After equilibration with excess ascorbate the solution potential is approx. +60 mV [33] and the electronic absorbance is changed. A smaller, broader Soret band and two small sharp peaks in the α-/β-band are indicative of approx. 13% heme reduction. Thus, the redox properties of PioA solutions are comparable to those of the protein adsorbed on conductive metal oxide electrodes ( figure 4).
For certain ligand sets Fe(III), but not Fe(II), hemes display ligand-to-metal charge-transfer bands between 600 and 750 nm at energies indicative of the chemical nature of their axial ligands [34]. In this spectral window the difference spectrum ( figure 5(B)) for ascorbate reduced minus oxidised PioA reveals two negative features. The trough centred at 630 nm is indicative of reduction of high-spin Fe(III) heme(s) most likely having His/H 2 O ligation. The trough centred at 700 nm is indicative of reduction of low-spin Fe(III) heme(s) with His/Met ligation. We conclude that oxidised PioA contains His/H 2 O and His/Met ligated hemes that are (partially) reduced by addition of ascorbate.
Addition of an excess of the strong reducing agent dithionite produces a solution potential of approx. −500 mV [35] and spectral features indicative of fully reduced PioA ( figure  5(A)). The Soret band maximum is at 419 nm and well-defined α-/β-band maxima are present at 552 and 523 nm respectively. In the difference spectrum for dithionite-reduced minus oxidised PioA no clear features were distinguished above 600 nm ( figure 5(B)). Instead a broad negative feature typical for the reduction of low-spin His/His ligated hemes is dominant.

Discussion
Multiheme cytochromes have evolved to move electrons across cell membranes and extracellular structures over distances exceeding several microns [36]. These remarkable properties have found application in the production of electricity by microbial fuel cells [37] and value-added chemicals during microbial electrosynthesis [38][39][40] where electrons may flow out from, or into, bacteria. Interest in harnessing the electrical properties of the purified multiheme cytochromes is also increasing. Their conductance can rival that of organic semiconductors and they offer renewable, readily engineered and flexible materials that electronically interface live cells and synthetic materials [36,41]. For these reasons, and to understand the determinants of biological electron transfer in diverse microorganisms, it is of interest to resolve structural and electronic properties of multiheme cytochromes from different bacterial species. Insights into these structure-function relationships are readily available from multi-disciplinary studies that combine electrochemical, spectroscopic and structural methods, e.g. [7,9,26,42,43]. In this work we used such an approach to provide the first detailed account of the  structure-function relationship for PioA. We find that PioA, which has evolved to transfer electrons across the bacterial outer membrane and into R. palustris TIE-1, differs in several properties from the homologous MtrA protein, which has evolved to transfer electrons out from Shewanella species.
The relatively well-studied outer membrane spanning MtrA protein of Shewanella species has His/His ligation of all ten-hemes [11]. By contrast this study has revealed His/Met and His/H 2 O ligated hemes in the outer membrane spanning PioA cytochrome of R. palustris TIE-1. Similarly ligated hemes are also present in the nonaheme cytochrome  [45] and edited in Jalview [46]. c-Type heme binding motifs are highlighted in gold and labelled based upon their position within the amino acid sequence. Known distal histidines of MtrA [11] and predicted distal histidines of MtoA and PioA are highlighted in blue. Predicted distal methionines of PioA are shown in red. The corresponding heme of the distal ligands, as determined from the structure of MtrA, are given in parentheses. (B) Homology model of PioA produced by Phyre 2 [47] using MtrA from S. baltica OS185 as a template [11]. The protein is shown in blue cartoon representation and the hemes are shown in yellow stick representation. Methionines which may potentially form distal ligands to Hemes 2, 3 and 7 are labelled and shown in pink stick representation.
OcwA that participates in extracellular electron transfer by the Gram-positive Thermincola potens JR [32]. MtrA and PioA have high sequence similarity, including the distribution of ten CxxCH c-heme binding motifs ( figure 6(A)). Combining this information with the x-ray structure of S. baltica OS185 MtrA [11] we are able to present a structural model for PioA (figure 6(B)) that provides molecular-level insight into the origin of differences between the MtrA and PioA cytochromes. The model has a chain of ten hemes spanning the longest dimension of PioA ( figure 6(B)). The hemes are arranged in order of the CxxCH binding motifs to which they are attached, such that the chain extends from N-terminal Heme 1 to Cterminal Heme 10. Neighbouring hemes sit in close proximity and alternate in having configurations of perpendicular and parallel porphyrin ring planes. The model is validated by hydrodynamic modelling of MtrA (90 Å × 39 Å × 45 Å) using SOMO [44] that predicts a sedimentation coefficient of 2.85 S, a value similar to that measured here for PioA (2.94 S). In addition, the MtrA model has a f /f 0 ratio of 1.30, close to the value of 1.33 measured for PioA.
PioA hemes receive proximal ligation by His of the corresponding CxxCH c-heme binding motifs. Of the ten distal His ligands identified in the crystal structure of S. baltica MtrA it is significant that only seven are conserved in PioA ( figure 6(A)). These conserved His residues are the most likely distal ligands of the corresponding PioA sites, namely, Hemes 1, 4, 5, 6, 8, 9 and 10. The distal His ligands to Hemes 2, 3 and 7 are not conserved and Met is found in place of the corresponding residues, at positions 382, 292 and 417 respectively (figure 6). Thus, Hemes 2, 3 and/or 7 are candidates for the His/Met ligated PioA heme(s) identified in this study. We note that for Heme 7, the predicted distal ligand of Met417 is preceded by His416 which could also ligate this heme. All ten distal His ligands of MtrA are conserved in the sequence of another MtrA-family member, MtoA of S. lithotrophicus ( figure 5(A)). Like MtrA and PioA, MtoA is associated with the bacterial outer membrane [9]. Like PioA, and in contrast to MtrA, MtoA naturally participates in electron uptake following Fe(II) oxidation ( figure 1(A)). Thus, there is no indication that His/Met ligation alone distinguishes decaheme cytochromes with a primary role passing electrons into, rather than out from, bacteria. Nevertheless, it is clear that His/Met ligation contributes to PioA having redox activity at potentials more positive than MtoA and MtrA (figure 7). It is possible that the His/Met ligated PioA hemes store electrons from Fe(II) oxidation prior to delivery to the periplasmic PioC protein (reduction potential ≈ +450 mV [48]) that is proposed [48] as the immediate donor to the reaction centre located in the inner membrane (figure 1(A) right). Full details of the electron transfer pathway and enzymology of carbon-fixation in R. palustris TIE-1 can be found in the studies of Guzman et al [12] and Bird et al [48].
The crystal structure of the S. baltica MtrA:MtrB complex shows MtrA Heme 10 presented on the cell surface and Hemes 1 and 2 presented to the periplasm [11]. If PioB wraps PioA in a similar manner, which seems likely, there will be little opportunity for the His/Met ligated hemes to participate in direct electron exchange with extracellular redox partners, soluble Fe(II) or electrodes. If Heme 10 is presented to the external environment it is tempting to suggest His/H 2 O ligation at this site to allow close approach of soluble Fe(II) complexes that may facilitate their oxidation. From sequence analysis, another candidate for His/H 2 O ligation is Heme 7. This species could exist as an intermediate if exchange of His416 and Met417 as distal ligands occurs. We note that the MtrA hemes have reduction potentials between approximately 0 and −400 mV (figure 7 [7]). Thus, we predict very little reduction of MtrA will occur when that protein is equilibrated with excess of the mild-reductant ascorbate. Because the MtrA crystal structure defines ten hemes with His/His ligation, we do not expect to see charge transfer bands indicative of His/Met or His/water ligated hemes in the electronic absorbance of MtrA.
The properties of PioA revealed in this study are significant for anticipating the corresponding features of the PioAhomologs predicted in a number of other species (e.g. Figure 1 and figure S1). A first defining feature of the PioA-family is cleavage of approximately 240 residues from the N-terminus that results in mature decaheme cytochromes of ≈ 40 kDa. This is shown here, and previously [19], for PioA of R. palustris TIE-1 and has been reported for the PioA proteins of R. vannielii DSM 162 and R. udaipurense JA643 [19]. Sequence analysis shows the potential for His/Met ligation of Heme 2 in decaheme PioA homologs from over 30 different bacterial genera ( figure S7). The adjacent His-Met pair of amino acids at the sequence position corresponding to the distal ligand of Heme 7 is also conserved in these homologs. His/Met ligation of Heme 3 is less common and appears to be primarily associated with PioA homologs from purple non-sulphur bacteria belonging to the alphaproteobacteria class. Thus, we propose the presence of His-Met ligated hemes is a second defining feature of the PioA-family that distinguishes them from the more widely studied MtrA-family.
From the perspective of biotechnology our results are significant in expanding knowledge of the molecular basis of microbial electrochemistry [5,6,[37][38][39][40]49]. The well-studied MtrA proteins have a natural role in electron export from Shewanella species and bacterial oxidation at anodes ( figure 1(A) left). By contrast PioA from R. palustris TIE-1 has evolved to allow electron uptake for bacterial light-driven carbon fixation ( figure 1(A) right). This process provides inspiration for strategies to synthesize valuable chemicals while reducing our reliance on fossil reserves and removing a potent greenhouse gas. R. palustris TIE-1 is genetically tractable [12,19]. However, to the best of our knowledge it has not yet been engineered for microbial electrosynthesis through lightdriven CO 2 -reduction. Such engineering is likely to require that electron transfer from cathodes to photosynthetic reaction centres is uncoupled from downstream pathways that are naturally optimised to use the photoenergised electrons for biomass production. Such an approach could circumvent the low current densities displayed by R. palustris TIE-1 which are typical for microbial electrosynthesis and presumed to be limited by the rate of biomass production [12,[16][17][18]38].
An alternate approach to enhanced microbial electrosynthesis would engineer an efficient conduit for electron uptake into a chassis microorganism developed for the desired chemical synthesis. The properties of PioA described here provide the first molecular details necessary for rational engineering of such a conduit in bacterial outer membranes.

Bacterial strains, plasmids and strains
The gene encoding PioA from R. palustris TIE-1 (1623 bp, Rpal_0817) was codon optimised for expression in S. oneidensis MR-1 and additional base pairs introduced to encode for a C-terminal Strep II tag (WSHPQFEK) to aid purification. Additional base pairs encoding a stop codon and a ribosome binding site were included at the 5 ′ end of the gene to aid overexpression when subcloned into the pBAD202/D-TOPO expression vector. The corresponding gene was amplified from plasmid pEX-K4_pioA (Eurofins Genomics) and cloned into pBAD202 utilizing a directional TOPO cloning kit (Invitrogen). The resulting vector, pBAD202_pioA, was transformed by electroporation into Shewanella oneidensis strain MR-1 and a variant lacking the mtr operon (LS527) [9] to give recombinant strains MR-1_pioA and ∆mtr_pioA, respectively. Full details of the gene sequence and the primers for PCR amplification are provided in the Supporting Information (figure S2).
After addition of SigmaFAST protease inhibitor, 1 mM dithiothreitol and DNase the cells were lysed by three passes through a French Press (16 000 psi). After addition (0.1 % by volume) of biotin blocking buffer (IBA Solutions for Lifesciences), cell debris and unbroken cells were pelleted by centrifugation (30 min, 5500 × g, 4 • C). The supernatant was recovered and subjected to centrifugation (60 min, 185 000 × g, 4 • C) to pellet the membrane fraction. Purification of PioA was from the soluble fraction (supernatant) and was monitored by electronic absorbance spectroscopy and SDS-PAGE; for the latter, proteins were visualized by Coomassie stain and peroxidaselinked heme stain employing 3-3 ′ -5-5 ′ tetramethylbenzidine. Soluble cell lysate was applied to a 5 ml Strep-Tactin XT Superflow Column (IBA Solutions for Lifesciences) equilibrated with Buffer A. After washing with Buffer A, bound protein was eluted with 40 mM biotin in Buffer A and applied to a Superose 6 Increase 10/300GL preparative grade gel filtration column equilibrated with 50 mM HEPES, 300 mM NaCl, pH 8.0. Eluted protein (A 409 /A 280 = 4.7) was concentrated and exchanged into 50 mM HEPES, 300 mM NaCl, pH 7.4 (Buffer B) using a centrifugal concentrator (3 kDa MWCO, PES, Pierce™). Aliquots of the purified protein were flash-frozen in liquid nitrogen and stored at −80 • C until required.

Biochemical analyses
LC-MS was performed as described previously [50] with PioA (typically 1 mg ml −1 ) diluted with formic acid (0.1% v/v) and acetonitrile (2% v/v) prior to analysis. Thiol(ate) was quantified using the Measure-iT™ Thiol Assay kit (Invitrogen) following the manufacturer's protocol with samples incubated at 37 • C for 30 min before spectroscopy; reduced glutathione produced the calibration curve and the fluorescence intensity at 517 nm was quantified for excitation at 494 nm. Protein concentration (mg ml −1 ) was quantified with BCA reagent (BioRad) using bovine serum albumin as the standard. Pyridine-hemochrome analysis was performed as described [24].

Analytical ultracentrifugation
Sedimentation equilibrium and sedimentation velocity were performed using a Beckman Optima XLA-I analytical ultracentrifuge equipped with scanning absorbance optics. Measurements were performed in Buffer B for which the density and viscosity were calculated as 1.0144 g ml −1 and 1.0611 × 10 −2 P respectively using Sedenterp software [25]. A partial specific volume (0.7347 ml g −1 ) was calculated from the amino acid sequence of truncated PioA using the utility software in Ultrascan II software [51]. Sedimentation equilibrium was performed at 20 • C using speeds of 15 k, 18 k, and 21 k rpm with absorbance profiles recorded at 409 nm (0.5 µM PioA) or 520 nm (5 µM PioA). Sedimentation equilibrium profiles were analysed with the programme Ultrascan II.
Sedimentation velocity was conducted at 20 • C and 40 k rpm with 0.8 µM PioA in 50 mM HEPES, 300 mM NaCl, pH 7.4. Absorbance was recorded at 409 nm every 2 min. Following both experiments, the samples were removed and SDS-PAGE gels stained for heme showed no evidence for PioA degradation. Data were fitted using the c(s) distribution analysis in the software SEDFIT [52]. The molecular weight M of PioA was obtained from the Svedberg equation,  [25], where S is the PioA sedimentation coefficient; D the PioA diffusion coefficient;ν the PioA partial specific volume, ρ the solvent buffer density, R the gas constant and T the temperature.

Electrochemistry
Spectro-potentiometry and cyclic voltammetry were performed by direct protein electrochemistry using threeelectrode cell configurations and an Autolab PGSTAT30 potentiostat (EcoChemie) controlled by NOVA software. For cyclic voltammetry the cell was inside a Faraday cage situated in a N 2 -chamber as previously described [53]. Potentiometric titration of PioA monitored by electronic absorbance spectroscopy was performed with the protein adsorbed on optically transparent mesoporous nanocrystalline SnO 2 electrodes. Electrodes were prepared by covering them with ice-cold solutions of PioA (17 µM) and the coadsorbate neomycin (25 mM) in Buffer B. After approximately 20 min the electrode was rinsed with Buffer B, to remove loosely-bound material, and mounted in an optical cuvette filled with anaerobic Buffer B and fitted with reference and counter electrodes as previously described [26]. Adsorbed PioA at ambient temperature was equilibrated at defined potentials and the electronic absorbance recorded. Spectral quality was optimised by mounting an equivalent electrode, lacking adsorbed protein, in the reference beam to minimise contributions from light scattering by the electrode. Protein film cyclic voltammetry was performed with PioA (17 µM) and the coadsorbate neomycin (25 mM) adsorbed on hierarchical indium tin oxide electrodes (750 nm macropore size, ∼50 nm mesoporous features, 20 µm film thickness, 0.25 cm 2 geometrical surface area [28,42]) placed in solutions at 20 • C containing 300 mM NaCl and 50 mM of either Bis-Tris (pH 6.4), HEPES (pH 7.4), or CHES (pH 9.4) for the indicated solution pH.

Bioinformatics
A BLAST [54] search was utilized to identify decaheme homologs of PioA, resulting in 773 protein sequences being retrieved. CxxCH motifs and their sequence locations were identified utilizing ScanProsite [55]. Signal peptides and predicted cleavage sites were identified utilizing SignalP 5.0 [56]. Sequences were aligned and phylogenetic trees calculated utilizing Clustal Omega [57]. Phylogenetic trees/Cladograms were formatted and protein features mapped using iTOL [58].
BB/P01819X, BB/S002499 to J N B and T A C and a Norwich Research Park Biosciences Doctoral Training Partnership Ph.D. studentship to S E H P) and Engineering and Physical Sciences Research Council (DTA Ph.D. studentship to K P S). D L is grateful to the Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO-CIC) and the Chinese Collaborative Academic Training Program for Post-doctoral Fellows. J N B acknowledges a Royal Society Leverhulme Trust Senior Research Fellowship.