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Probing domain mobility in a flavocytochrome.

Rothery EL, Mowat CG, Miles CS, Mott S, Walkinshaw MD, Reid GA, Chapman SK
Biochemistry 43 4983-9 (2004)
Cited: 10 times
EuropePMC logo PMID: 15109257

Abstract

The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release.

Articles - 1q9i mentioned but not cited (1)

  1. Hidden relationships between metalloproteins unveiled by structural comparison of their metal sites. Valasatava Y, Andreini C, Rosato A. Sci Rep 5 9486 (2015)


Reviews citing this publication (2)

  1. Catalytic mechanisms of complex II enzymes: a structural perspective. Iverson TM. Biochim Biophys Acta 1827 648-657 (2013)
  2. Direct Electron Transfer of Enzymes Facilitated by Cytochromes. Ma S, Ludwig R. ChemElectroChem 6 958-975 (2019)

Articles citing this publication (7)

  1. Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state. Huang LS, Shen JT, Wang AC, Berry EA. Biochim Biophys Acta 1757 1073-1083 (2006)
  2. A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina. Pankhurst KL, Mowat CG, Rothery EL, Hudson JM, Jones AK, Miles CS, Walkinshaw MD, Armstrong FA, Reid GA, Chapman SK. J Biol Chem 281 20589-20597 (2006)
  3. A threonine on the active site loop controls transition state formation in Escherichia coli respiratory complex II. Tomasiak TM, Maklashina E, Cecchini G, Iverson TM. J Biol Chem 283 15460-15468 (2008)
  4. Molecular basis of maintaining an oxidizing environment under anaerobiosis by soluble fumarate reductase. Kim S, Kim CM, Son YJ, Choi JY, Siegenthaler RK, Lee Y, Jang TH, Song J, Kang H, Kaiser CA, Park HH. Nat Commun 9 4867 (2018)
  5. New crystal forms of the integral membrane Escherichia coli quinol:fumarate reductase suggest that ligands control domain movement. Starbird CA, Tomasiak TM, Singh PK, Yankovskaya V, Maklashina E, Eisenbach M, Cecchini G, Iverson TM. J Struct Biol 202 100-104 (2018)
  6. Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR. Pessanha M, Rothery EL, Louro RO, Turner DL, Miles CS, Reid GA, Chapman SK, Xavier AV, Salgueiro CA. FEBS Lett 578 185-190 (2004)
  7. Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella. Paquete CM, Saraiva IH, Louro RO. Biochim Biophys Acta 1837 717-725 (2014)


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