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. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.
Pessanha
Miguel
M
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal.
Rothery
Emma L
EL
Louro
Ricardo O
RO
Turner
David L
DL
Miles
Caroline S
CS
Reid
Graeme A
GA
Chapman
Stephen K
SK
Xavier
António V
AV
Salgueiro
Carlos A
CA
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D020592:Shewanella / Q000737:chemistry / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D006418:Heme; C112400:flavocytochrome c3; D013385:Succinate Dehydrogenase