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Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS I04-Macromolecular Crystallography	A cysteine pair controls flavin reduction by extracellular cytochromes during anoxic/oxic environmental transitions Michael P. Norman , Marcus J. Edwards , Gaye F. White , Joshua A. J. Burton , Julea N. Butt , David J. Richardson , Ricardo O. Louro , Catarina M. Paquete , Thomas A. Clarke Mbio DOI: 10.1128/mbio.02589-22 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment.	Jan 2023
I04-Macromolecular Crystallography	Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme Jessica H. Van Wonderen , Katrin Adamczyk , Xiaojing Wu , Xiuyun Jiang , Samuel E. H. Piper , Christopher R. Hall , Marcus J. Edwards , Thomas A. Clarke , Huijie Zhang , Lars J. C. Jeuken , Igor V. Sazanovich , Michael Towrie , Jochen Blumberger , Stephen R. Meech , Julea N. Butt Proceedings Of The National Academy Of Sciences 118 DOI: 10.1073/pnas.2107939118 Diamond Proposal Number(s): [25108] Open Access Show Abstract ▼ Abstract: 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.	Sep 2021
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I23-Long wavelength MX	The crystal structure of a biological insulated transmembrane molecular wire Marcus Edwards , Gaye F. White , Julea N. Butt , David J. Richardson , Thomas A. Clarke Cell DOI: 10.1016/j.cell.2020.03.032 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand β-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.	Apr 2020
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer Marcus Edwards , Gaye F. White , Michael Norman , Alice Tome-Fernandez , Emma Ainsworth , Liang Shi , Jim K. Fredrickson , John M. Zachara , Julea N. Butt , David J. Richardson , Thomas A. Clarke Scientific Reports 5 DOI: 10.1038/srep11677 PMID: 26126857 Diamond Proposal Number(s): [7641] Open Access Show Abstract ▼ Abstract: Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.	Jul 2015
I03-Macromolecular Crystallography	Characterization of MtoD from Sideroxydans lithotrophicus: a cytochrome c electron shuttle used in lithoautotrophic growth Christopher Beckwith , Marcus Edwards , Matthew Lawes , Liang Shi , Julea N. Butt , David J. Richardson , Thomas A. Clarke Frontiers In Microbiology 6 DOI: 10.3389/fmicb.2015.00332 PMID: 25972843 Diamond Proposal Number(s): [7641] Open Access Show Abstract ▼ Abstract: The autotrophic Sideroxydans lithotrophicus ES-1 can grow by coupling the oxidation of ferrous iron to the reduction of oxygen. Soluble ferrous iron is oxidised at the surface of the cell by an MtoAB porin-cytochrome complex that functions as an electron conduit through the outer membrane. Electrons are then transported to the cytoplasmic membrane where they are used to generate proton motive force (for ATP synthesis) and NADH for autotrophic processes such as carbon fixation. As part of the mtoAB gene cluster, S. lithotrophicus also contains the gene mtoD that is proposed to encode a cytochrome c protein. We isolated mtoD from a Shewanella oneidensis expression system where the mtoD gene was expressed on a pBAD plasmid vector. Biochemical, biophysical and crystallographic characterisation of the purified MtoD revealed it as an 11 kDa monomeric protein containing a single heme. Sequence and structural alignment indicated that MtoD belonged to the class-1 cytochrome c family and had a similar fold to ferricytochrome c552 family, however the MtoD heme is bis-histidine coordinated and is substantially more exposed than the hemes of other family members. The reduction potential of the MtoD heme at pH 7 was +155 mV vs. Standard Hydrogen Electrode, which is approximately 100 mV lower than that of mitochondrial cytochromes c. Consideration of the properties of MtoD in the context of the potential respiratory partners identified from the genome suggests that MtoD could associate to multiple electron transfer partners as the primary periplasmic electron shuttle.	Apr 2015
I24-Microfocus Macromolecular Crystallography	The X-ray crystal structure of Shewanella oneidensis OmcA reveals new insight at the microbe-mineral interface Marcus J. Edwards , Nanakow A. Baiden , Alexander Johs , Stephen J. Tomanicek , Liyuan Liang , Liang Shi , Jim K. Fredrickson , John M. Zachara , Andrew J. Gates , Julea N. Butt , David J. Richardson , Thomas A. Clarke Febs Letters 588 (10), 1886 - 1890 DOI: 10.1016/j.febslet.2014.04.013 Diamond Proposal Number(s): [9475] Show Abstract ▼ Abstract: he X-ray crystal structure of Shewanella oneidensis OmcA, an extracellular decaheme cytochrome involved in mineral reduction, was solved to a resolution of 2.7 Å. The four OmcA molecules in the asymmetric unit are arranged so the minimum distance between heme 5 on adjacent OmcA monomers is 9 Å, indicative of a transient OmcA dimer capable of intermolecular electron transfer. A previously identified hematite binding motif was identified near heme 10, forming a hydroxylated surface that would bring a heme 10 electron egress site to ∼10 Å of a mineral surface.	May 2014
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	A new crystal structure of the bifunctional antibiotic simocyclinone D8 bound to DNA gyrase gives fresh insight into the mechanism of inhibition Stephen Hearnshaw , Marcus Edwards , Clare Stevenson , David Lawson , Anthony Maxwell Journal Of Molecular Biology DOI: 10.1016/j.jmb.2014.02.017 PMID: 24594357 Diamond Proposal Number(s): [1219, 7641] Open Access Show Abstract ▼ Abstract: Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~ 1000-fold weaker than the interaction with GyrA.	Mar 2014
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	The crystal structure of the extracellular 11-heme cytochrome UndA reveals a conserved 10-heme motif and defined binding site for soluble iron chelates Marcus J. Edwards , Andrea Hall , Liang Shi , James K. Fredrickson , John M. Zachara , Julea N. Butt , David J. Richardson , Thomas A. Clarke Structure 20 (7), 1275-1284 DOI: 10.1016/j.str.2012.04.016 PMID: 22682743 Diamond Proposal Number(s): [505494] Show Abstract ▼ Abstract: Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure has also been crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.	Jul 2012
I02-Macromolecular Crystallography	Structure of a bacterial cell surface decaheme electron conduit Thomas A. Clarke , Marcus J. Edwards , Andrew J. Gates , Andrea Hall , Gaye F. White , Justin Bradley , Catherine L. Reardon , Liang Shi , Alexander S. Beliaev , Matthew J. Marshall , Zheming Wang , Nicholas J. Watmough , James K. Fredrickson , John M. Zachara , Julea N. Butt , David J. Richardson Proceedings Of The National Academy Of Sciences 108 (23), 9384-9389 DOI: 10.1073/pnas.1017200108 PMID: 21606337 Show Abstract ▼ Abstract: 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 β-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.	Jun 2011

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