Limits...
Protein-protein interaction regulates the direction of catalysis and electron transfer in a redox enzyme complex.

McMillan DG, Marritt SJ, Firer-Sherwood MA, Shi L, Richardson DJ, Evans SD, Elliott SJ, Butt JN, Jeuken LJ - J. Am. Chem. Soc. (2013)

Bottom Line: Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase.Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7.These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Protein-protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein-protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis . We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis . Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein-protein interactions.

Show MeSH
CVs (10 mV/s) of a SSM on a gold electrode modifiedwith cholesteroltethers. The SSM was formed with CymA proteoliposomes (90:10 POPC:cardiolipin;1% (w/w) CymA; 1% (w/w) MQ-7). (A) CVs (a) before and (b) after additionof 1 mM potassium ferricyanide. (B) CVs (a) before and (b,c) afteraddition of (b) 1 mM sodium dithionite and (c) 1 mM sodium dithionite/10μM HQNO.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3823026&req=5

fig3: CVs (10 mV/s) of a SSM on a gold electrode modifiedwith cholesteroltethers. The SSM was formed with CymA proteoliposomes (90:10 POPC:cardiolipin;1% (w/w) CymA; 1% (w/w) MQ-7). (A) CVs (a) before and (b) after additionof 1 mM potassium ferricyanide. (B) CVs (a) before and (b,c) afteraddition of (b) 1 mM sodium dithionite and (c) 1 mM sodium dithionite/10μM HQNO.

Mentions: The experiments described above reveal that the CymA-Fcc3 complex is able to oxidize MQ-7 in SSMs. This is in starkcontrastto our previous studies of CymA in contact with MQ-7 containing liposomeswhere only MQ-7 reduction was observed.11 To establish whether Fcc3 is responsible for the changein the catalytic bias of CymA, the activity of CymA in the membrane-modifiedelectrodes was measured in the absence of Fcc3 using chemicalreductants and oxidants (Figure 3). Additionof ferricyanide had almost no effect on the voltammogram (Figure 3A). Electronic absorbance spectroscopy demonstratesthat solutions of reduced CymA are rapidly oxidized by ferricyanide(not shown). As a consequence, the absence of a reductive signal inthe CV confirms that CymA without Fcc3 is unable to oxidizeMQ-7. The reduction potential of ferricyanide (+420 mV) is higherthan that of fumarate (−30 mV), indicating that MQ-7 oxidationby the CymA-Fcc3 complex is not driven solely by thermodynamics.Instead, formation of the long-lived CymA-Fcc3 complexshifts the catalytic bias of CymA toward MQ-7 oxidation. When, insteadof ferricyanide, dithionite is added to the SSM, a catalytic oxidationsignal appears that is inhibited by addition of HQNO. This confirmsour previous findings, that CymA is only able to reduce MQ-7 in theabsence of Fcc3.


Protein-protein interaction regulates the direction of catalysis and electron transfer in a redox enzyme complex.

McMillan DG, Marritt SJ, Firer-Sherwood MA, Shi L, Richardson DJ, Evans SD, Elliott SJ, Butt JN, Jeuken LJ - J. Am. Chem. Soc. (2013)

CVs (10 mV/s) of a SSM on a gold electrode modifiedwith cholesteroltethers. The SSM was formed with CymA proteoliposomes (90:10 POPC:cardiolipin;1% (w/w) CymA; 1% (w/w) MQ-7). (A) CVs (a) before and (b) after additionof 1 mM potassium ferricyanide. (B) CVs (a) before and (b,c) afteraddition of (b) 1 mM sodium dithionite and (c) 1 mM sodium dithionite/10μM HQNO.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3823026&req=5

fig3: CVs (10 mV/s) of a SSM on a gold electrode modifiedwith cholesteroltethers. The SSM was formed with CymA proteoliposomes (90:10 POPC:cardiolipin;1% (w/w) CymA; 1% (w/w) MQ-7). (A) CVs (a) before and (b) after additionof 1 mM potassium ferricyanide. (B) CVs (a) before and (b,c) afteraddition of (b) 1 mM sodium dithionite and (c) 1 mM sodium dithionite/10μM HQNO.
Mentions: The experiments described above reveal that the CymA-Fcc3 complex is able to oxidize MQ-7 in SSMs. This is in starkcontrastto our previous studies of CymA in contact with MQ-7 containing liposomeswhere only MQ-7 reduction was observed.11 To establish whether Fcc3 is responsible for the changein the catalytic bias of CymA, the activity of CymA in the membrane-modifiedelectrodes was measured in the absence of Fcc3 using chemicalreductants and oxidants (Figure 3). Additionof ferricyanide had almost no effect on the voltammogram (Figure 3A). Electronic absorbance spectroscopy demonstratesthat solutions of reduced CymA are rapidly oxidized by ferricyanide(not shown). As a consequence, the absence of a reductive signal inthe CV confirms that CymA without Fcc3 is unable to oxidizeMQ-7. The reduction potential of ferricyanide (+420 mV) is higherthan that of fumarate (−30 mV), indicating that MQ-7 oxidationby the CymA-Fcc3 complex is not driven solely by thermodynamics.Instead, formation of the long-lived CymA-Fcc3 complexshifts the catalytic bias of CymA toward MQ-7 oxidation. When, insteadof ferricyanide, dithionite is added to the SSM, a catalytic oxidationsignal appears that is inhibited by addition of HQNO. This confirmsour previous findings, that CymA is only able to reduce MQ-7 in theabsence of Fcc3.

Bottom Line: Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase.Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7.These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Protein-protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein-protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis . We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis . Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein-protein interactions.

Show MeSH