Comparative structure-potentio-spectroscopy of the Shewanella outer membrane multiheme cytochromes

Many species of bacteria can generate energy in the anoxic subsurface by directly coupling intracellular oxidative reactions to the reduction of extracellular metal oxides. Coupling these processes requires electron transfer networks that extend from the inside of the cell, across the outer membrane to the extracellular terminal electron acceptors. The best described of these networks is from Shewanella oneidensis MR-1, where four structures of outer membrane multiheme cytochromes (OMMCs) have been determined. These OMMCs contain 10–11 bis -histidine-ligated c-type hemes and are directly involved in the reduction of iron and manganese oxides at the cell surface. The heme ligands for some of these structures have been characterised using electron paramagnetic resonance (EPR), the redox properties have been mapped by protein film electrochemistry (PFE) and more recently molecular dynamic simulations have been used to obtain microscopic redox potentials for individual heme groups. This review maps these different experimental techniques onto the structures, providing insight into the intramolecular electron transfer pathways of OMMCs, revealing future directions for study.


Introduction
Many bacteria can couple heterotrophic oxidation of organic matter to the reduction of terminal oxidants that substitute for molecular oxygen, a process commonly referred to as anaerobic respiration. This physiological property of bacteria is facilitated by oxidoreductase networks that oxidise quinol in the cytoplasmic membrane and relay the liberated electrons to specific terminal electron acceptor(s) [1] . The majority of Shewanella spp. have been shown to utilise insoluble non-diffusing terminal respiratory oxidants, namely soluble and insoluble transition metals [2,3] . Biogenic reduction of minerals is a link between carbon cycling and geochemical cycles that affect mineral formation [4,5] . Mineral reduction also affects redox processes such as methanogenesis and sulphate reduction in soil and aquatic niches [6,7] , and the dissemination of many mineral-adsorbed molecules [8][9][10][11] . The final enzyme-substrate electron transfer step at the microbe-mineral interface has been shown to be dependent on the expression of multiheme cytochromes associated with the Shewanella outer membrane [12,13] . MtrA and MtrD are decaheme cytochromes that form putative porin-cytochrome modules (with respective βbarrels MtrB and MtrE) to relay electrons from periplasmic reductants across the outer membrane [12,14,15] . Electrons exit the porin-cytochrome complexes into outer membrane multiheme cytochromes (OMMCs) on the surface of the cell [16,17] . This review compares the spectropotentiometric and structural data available for the four major clades of OMMCs that localise to the extracellular surface of the Shewanella outer bacterial membrane [18][19][20][21][22] .

Structural features of the Shewanella OMMC family
Shewanellacea genomes encode for multiple OMMCs with apparently similar functions. Bioinformatic analysis of Shewanella spp. shows that the minimal OMMCs encoded for are the decahemes MtrC and either OmcA or UndA [23] . MtrC has been extensively characterised for its affinity to the MtrAB complex and its canonical role as a mineral reductase [15,16] . MtrF has been identified as a component of the paralogous MtrDEF heterotrimer, which is up-regulated in aerobic S. oneidensis MR-1 biofilms [24] . A lipid anchor maintains OmcA's outer membrane localisation, and its mineral reduction activity is dependent on its association to MtrCAB [13,25,26] . A comprehensive gene knock-out study determined that the presence of either mtrC or omcA in the S. oneidensis genome maintained significant wild-type iron oxide reduction [13] . Known mineral-reducing Shewanella species that do not have omcA in their genomes encode for UndA, an undecaheme cytochrome indicated to have an analogous Crystal structures of MtrC [30] , MtrF [27] , OmcA [28] of Shewanella oneidensis MR-1 and UndA [29] of Shewanella sp. Strain HRCR-6. Polypeptide chains are shown in cartoon representation and coloured from blue (N-terminus) to red (C-terminus). Hemes are shown in stick representation with the iron atoms shown as orange spheres.
The molecular structures of representatives of each of the four OMMC clades in Shewanella have been determined: the decaheme MtrF, OmcA and MtrC of S. oneidensis and the undecaheme UndA of S. HRCR-6 ( Figure 1 ) [27-30 •• ] . All structures are formed of four domains, two multiheme domains that are flanked by two β-barrels with βstrands arranged in Greek key motifs. MtrF, OmcA and MtrC contain a conserved decaheme 'staggered cross' cofactor arrangement, with UndA containing an 11th heme that is inserted between hemes 6 and hemes 7 in the amino acid sequence. The staggered cross heme arrangement means there are four potential sites for electrons to enter and exit the structure, with two opposing ends of the cross pointing into the β-barrels and two exposed at the edges of the multiheme domains. The structures also reveal a conserved CX [8][9][10][11][12][13][14][15] C disulphide within the β-barrel of domain III. A second CX 2 -3 C disulphide is in UndA, OmcA and a subpopulation of MtrC located within the N-terminal β-barrel domain I; this CX 3 C motif is present in the amino acid sequence of MtrF but the putative disulphide bond was not resolved due to poor electron density in that domain. There is appreciable structural conservation of OMMC domains, as well as significant maintenance of heme positioning, despite less than 25% sequence homology between the least divergent clade members [28,30] .

The comparative spectropotentiometric properties of the OMMCs
The internal molecular landscape of the OMMCs is likely to lead to distinct energy landscapes and subsequently different redox properties of the proteins. Protein film electrochemistry (PFE) provides high-resolution potentiometric data of electrode-adsorbed protein [31] . The OMMCs are lipoproteins and it could be argued that protein in solution mimics their molecular environment best, although it is equally noted that molecular crowding at a densely packed membrane surface with an insoluble substrate may be better reproduced by adsorbing a protein to an electrode [32] . As such, the advantage of high-resolution data comes at the expense of a possibility that some proteins may have different potentiometric properties in solution than when surface adsorbed. The PFE of the four OMMCs provides very accurate, high-resolution characterisation of the cytochromes' oxidation state that highlights their different redox potential behaviour ( Figure 2 ) [27,33,34] . For each cytochrome a representative reducing and oxidising scan is plotted, where the respective negative and positive Faradaic responses correspond to the protein performing electron transfer to (positive response) and from (negative response) the working electrode during cyclic voltammetry (CV). Each OMMC voltammogram was successfully confirmed to have peaks that remained unchanged during rapid electrode rotation and a change of buffer, confirming adsorbed species were producing the voltammetric responses observed. The redox potentials of the OMMC are broadly similar, although MtrC contains a population of Protein film electrochemistry of extracellular cytochromes. Cyclic voltammetry of MtrC (black line), pH 7.6 [15] , MtrF (red line), pH 7.0 [27] , OmcA (grey dash), pH 7.4 [34] and UndA (blue dash), pH 7, measured at 30 mV/s in 25 mM HEPES, 100 mM NaCl.
hemes that are at a higher potential than the other OMMC and lacks the lower potential hemes present for the other OMMC. The hemes of MtrF cover a broad range of potentials, and includes a small population of hemes that correlate to approximately ≥−250 mV vs standard hydrogen electrode. Previously, the potential window of MtrC was shown to be higher than OmcA, with apparent midpoint potentials and window of −138 ± 275 mV for MtrC and −175 ± 300 mV for OmcA reported at pH 6 at 4 °C on graphite electrodes [35] . Despite the differences in potential, the overlapping potential windows of MtrC and OmcA indicate that reversible electron exchange between the two OMMC, in particular MtrC and OmcA, is possible.
EPR-monitored potentiometric titres provide information on specific heme populations, based on the spectroscopic resolution of heme populations that can be correlated to their respective OMMC crystal structure. Low spin, ferric heme has one unpaired 3 d valence electron; whereas ferrous heme has zero unpaired valence electrons. Thus, EPR potentiometric titrations can monitor the redox transformations of the OMMC c -type hemes from their resonating, oxidised S = 1 2 state to their EPRsilent reduced S = 0 state. To date, MtrC, MtrF and OmcA potentiometric data have been published [27,33] . The oxidised EPR spectra of MtrF and MtrC are characteristic of low spin (S = ½) ferric heme populations grouped into two clusters, denoted LS (i.e., low spin) or LGM (i.e., large g max ) ( Figure 3 ). Near-perpendicular bis -histidine ligand heme rings result in increased rhombicity of LGM signals which thus appear on the low field side of the LS signals at g 1 ≈ 3.7 − 3.1 [36] . The LS signals are rhombic signals (i.e., 3.1 > g 1 > 2.5) characteristic of low spin fer-

Figure 4
The structural arrangement of MtrF hemes. Hemes of domain II and IV are shown within blue and red boxes, respectively. Hemes with near-perpendicular bis -histidine ligation that would give LGM EPR signals ( ϕ b H ≥ 45 °) are shown in blue. Near-parallel bis-histidine ligated that would give LS1 EPR signals ( ϕ b H ≤ 45 °) are shown in red. Heme 10, that is likely to give an imidazolate histidine based on the crystal structure and generate an LS2 EPR signal, is shown in cyan. Each heme has assigned microscopic redox potentials that correspond to single-electron reduction of a fully oxidised MtrF. Microscopic redox potentials are from Wanatabe et al. (left [38] ) and Breuer et al. (right [39] ). The macroscopic heme potential determined for heme 10 is shown in bold [27] . Heme potentials values shown in volts.
ric hemes with decreased rhombicity, such as near-parallel bis -imidazole rings (i.e., g 1 ≈ 3.0 − 2.9). The EPR spectrum of OmcA contained a signal attributed to a population of high spin heme in addition to both LS and LGM signals [37] . However, the crystal structure of OmcA revealed that all 10 heme groups were low spin, suggesting that the observed high spin signal could be due to a small population of high spin hemes within the sample, possibly originating from denatured protein.
It is possible to tentatively assign resonance signals to specific structural heme populations in the oxidised OMMCs in conjunction with the bis-histidine ring dihedral angle ( ϕ b H ) data available from the published crystal structures ( Figure 4 ). The published EPR spectrum of MtrC contains three resonance populations, including LGM ( g 1 ≈ 3.2), and two low spin: LS1 ( g 1 ≈ 3.0) and LS2 ( g 1 ≈ 2.86) ( Figure 3 ). Spin quantitation was not reported in this study, and so resonance populations cannot be attributed to structural hemes although the propionate group of heme 6 may deprotonate its own distal His 500 ligand, producing the low potential LS2 signal. LS1 appears to be the dominant resonance population by g 1 peak height. However, the double integral of the LGM signal, that has a comparable g 1 peak height to the LS1 signal and must have a larger magnetic field coverage, is directly proportional to spin concentration and may be larger in MtrC. This would reflect the distribution of ϕ b H amongst MtrC's hemes: excluding the putative LS2 heme 6, there are four LGM candidate hemes and six LS1 candidates.
Spin quantitation of the EPR spectrum of oxidised MtrF reported 7.86 spins per protein split into three different resonance signals. The quantitation of less than 10 spins per oxidised MtrF was attributed to possible dipolar resonance coupling between hemes [27] . The predominantly low ϕ b H of MtrF's hemes is reflected in the EPR spectrum, which accounts for five to six LS1 hemes of the six crystallographically observed (i.e., hemes 2, 4, 5, 6, 7 and 8; g 1,2, 3 = 3.0, 2.26, and 1.5) ( Figure 3 ). Two of the three near-perpendicular bis -histidine-ligated hemes (i.e., hemes 1, 3, 9; g 1,2, 3 = 3.25, 2.02, and 1.15) are accounted for in spin quantitation of MtrF's LGM resonance signal. The histidinate-ligated heme 10 is attributed to producing LS2 resonance ( g 1,2, 3 = 2.84, 2.31, and 1.63). The LS1 heme population of near-parallel hemes titrate over a broad potential window, 0 to −250 mV vs S.H.E., and the g 1 peak height dominated the oxidised EPR spectrum, further supporting the origin of the signal from multiple hemes. The broad g 1 = 3. 26 LGM signal is produced by near-perpendicular bis -histidine ligand pairs (hemes 1, 3, or 9) although signal quantitation only accounts for two of these LGM hemes titrating between −100 and −260 mV. The LS2 signal (i.e., g 1 = 2.84) originating from ferric heme with an imidazolate ligand amounts to a single heme (i.e., heme 10), the only signal remaining at −260 mV. As such heme 10 is putatively the lowest potential heme of MtrF. This correlates well with the PFV, which also identified a low potential heme at −250 mV.
Large-scale molecular dynamic simulations have attempted to resolve the electron transport network through the ten hemes of the MtrF molecular structure. Microscopic redox potentials for the single-electron reduction of each individual heme, with the other nine hemes in the oxidised states (i.e., oxidised MtrF) have been reported by two groups [38,39] ( Figure 4 ). Breuer et al. [39] suggested that the potentials of the heme pairs across the two pentaheme domains were similar, allowing for bidirectional electron transfer pathways across MtrF. Watanabe et al. [38] alternatively proposed that the heme potentials in domain IV were lower than in domain II, suggesting a downhill electron transfer path from domain IV to domain II. Both studies identified the heme potentials in hemes 2 and 7 as being suitable for electron transfer to flavin groups and suggested heme 9 as being the lowest potential heme group, as opposed to heme 10 that was inferred from the crystal structure. This may be due to the challenges of accurately modelling a low-resolution MtrF structure, and it will be interesting to compare similar studies on the high-resolution MtrC structure in the future.

The interaction of flavins and OMMCs
S. oneidensis secretes both riboflavin and flavin mononucleotide (FMN) and these secreted flavins have a substantial impact on the ability of S. oneidensis to reduce Fe(III) oxides [40] . One hypothesis is that both riboflavin and FMN function as soluble redox mediators that facilitate electron exchange between S. oneidensis and solid metal oxides [41] . This is supported by studies that identified a bfe gene in S. oneidensis that was essential both for secretion of flavin into the extracellular medium and the ability of S. oneidensis to reduce Fe(III) oxides [42 • ] . As both bfe and mtr are important for extracellular electron transfer it is likely that flavins must interact with either directly or indirectly with extracellular OMMC on the cell surface. Dissociation constants of 29 μM and 255 μM between oxidised FMN and oxidised MtrC or OmcA, respectively, have been measured [43] . Given that the S. oneidensis extracellular FMN concentration does not exceed 1 μM during growth, these dissociation constants suggest a transient interaction between FMN and the OMMC. However, electrochemical and voltammetric studies on S. oneidensis biofilms generated on the surface of electrodes indicated that under anaerobic conditions MtrC associated with FMN to produce a semireduced flavin at the biofilm-flavin interface, suggesting the formation of a MtrC-FMN complex [44,45 • ] . These different results could be harmonised if the interaction between MtrC and FMN was different under aerobic and anaerobic respiratory conditions. Accordingly, a reversible transition of MtrC between cytochrome and flavocytochrome states that is controlled by the redox state of a conserved disulphide has been reported [30] . S. oneidensis strains that are unable to form the disulphide were severely compromised in their ability to grow aerobically, but not anaerobically, suggesting that the MtrC-FMN flavocytochrome may reduce oxygen and produce reactive oxygen species, so its formation must be closely regulated during life at oxic-anoxic interfaces. This indicates that the flavin must bind within the proximity of conserved disulphide and close enough to one or more heme groups to support electron transfer at catalytic rate. For OmcA mutation of the distal axial ligand of heme 7 in OmcA has been shown to lower the affinity of OmcA for FMN, indicating a similar interaction [46] . Different groups have utilised molecular dynamic simulations to identify riboflavin and FMN binding sites on OMMC [43,47 • ,48 • ] . Most recently two sites capable of binding riboflavin FMN binding sites were identified on OmcA close to hemes 5 and 7, while up to four possible binding sites were identified on MtrC close to hemes 1, 4, 7 and 9 [49 •• ] . The experimental data support the binding of two flavin molecules to OmcA, and only one to MtrC, so it is still unclear which of these MD simulations best supports the experimental data.

Conclusion
The four X-ray crystal structures of representative members of each of the four clades of OMMC published over the last 6 years have provided a molecular foundation for many research groups to study the electrochemical properties of these proteins, both experimentally and through theoretical models. Much remains to be understood though, notably the precise nature of flavin binding to the OMMCs and the structures of the complexes with which the OMMCs interact in order to receive electrons from inside the cell at the cell surface. Functional MtrCAB complexes that catalyses trans-outer membrane electron transfer in proteoliposomes have given some insight into this process, but further progress on a molecular understanding that might underpin further electrochemical insights will benefit from a high-resolution structure of the complex.