Tuning of functional heme reduction potentials in Shewanella fumarate reductases.

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria.

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate.

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR.

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems.

Octaheme tetrathionate reductase is a respiratory enzyme with novel heme ligation.

We have isolated a soluble cytochrome from Shewanella oneidensis that contains eight covalently attached heme groups and determined its crystal structure. One of these hemes exhibits novel ligation of the iron atom by the epsilon-amino group of a lysine residue, despite its attachment via a typical CXXCH motif. This heme is most likely the active site for tetrathionate reduction, a reaction catalyzed efficiently by this enzyme.

Probing domain mobility in a flavocytochrome.

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.

Histidine 61: an important heme ligand in the soluble fumarate reductase from Shewanella frigidimarina.

An examination of the X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L.

Thermodynamic characterization of a tetrahaem cytochrome isolated from a facultative aerobic bacterium, Shewanella frigidimarina: a putative redox model for flavocytochrome c3.

The facultative aerobic bacterium Shewanella frigidimarina produces a small c-type tetrahaem cytochrome (86 residues) under anaerobic growth conditions. This protein is involved in the respiration of iron and shares 42% sequence identity with the N-terminal domain of a soluble flavocytochrome, isolated from the periplasm of the same bacterium, which also contains four c -type haem groups. The thermodynamic properties of the redox centres and of an ionizable centre in the tetrahaem cytochrome were determined using NMR and visible spectroscopy techniques.