A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions
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 extracellula...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-02-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.02589-22 |
| _version_ | 1811163366523142144 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Michael P. Norman |
| collection | DOAJ |
| description | 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. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface. |
| first_indexed | 2024-04-10T06:43:46Z |
| format | Article |
| id | doaj.art-3ca985b053494e71b746ed11cbf4c2a4 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-10T06:43:46Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
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| spelling | doaj.art-3ca985b053494e71b746ed11cbf4c2a42023-02-28T14:06:24ZengAmerican Society for MicrobiologymBio2150-75112023-02-0114110.1128/mbio.02589-22A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental TransitionsMichael P. Norman0Marcus J. Edwards1Gaye F. White2Joshua A. J. Burton3Julea N. Butt4David J. Richardson5Ricardo O. Louro6Catarina M. Paquete7Thomas A. Clarke8Babraham Institute, Babraham Research Campus, Babraham, Cambridge, United KingdomSchool of Life Sciences, University of Essex, Colchester, United KingdomSchool of Biological Sciences and School of Chemistry, University of East Anglia, Norwich, United KingdomSchool of Biological Sciences and School of Chemistry, University of East Anglia, Norwich, United KingdomSchool of Biological Sciences and School of Chemistry, University of East Anglia, Norwich, United KingdomSchool of Biological Sciences and School of Chemistry, University of East Anglia, Norwich, United KingdomInstituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa, Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa, Oeiras, PortugalSchool of Biological Sciences and School of Chemistry, University of East Anglia, Norwich, United KingdomABSTRACT 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. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.https://journals.asm.org/doi/10.1128/mbio.02589-22peroxideflavoproteinflavinShewanella oneidensiscytochromecysteine |
| spellingShingle | 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 A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions mBio peroxide flavoprotein flavin Shewanella oneidensis cytochrome cysteine |
| title | A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions |
| title_full | A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions |
| title_fullStr | A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions |
| title_full_unstemmed | A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions |
| title_short | A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions |
| title_sort | cysteine pair controls flavin reduction by extracellular cytochromes during anoxic oxic environmental transitions |
| topic | peroxide flavoprotein flavin Shewanella oneidensis cytochrome cysteine |
| url | https://journals.asm.org/doi/10.1128/mbio.02589-22 |
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