Redox characterization of Geobacter sulfurreducens cytochrome c7: physiological relevance of the conserved residue F15 probed by site-specific mutagenesis.
The complete genome sequence of the delta-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c(7), isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with M(r) 9.6 kDa. This protein is involved in metal ion-reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related delta-proteobacterium Desulfuromonas acetoxidans (Dac(7)). In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G. sulfurreducens triheme cytochrome c(7) (PpcA). NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure [Pokkuluri, P. R., Londer, Y. Y., Duke, N. E. C., Long, W. C., and Schiffer, M. (2004) Biochemistry 43, 849-859] using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2. Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA. The results obtained showed that PpcA and Dac(7) have different redox properties: (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox-Bohr effect) in the physiological pH range, which is not observed with Dac(7). The differences observed in the redox behavior of PpcA and Dac(7) may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions. The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role.
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal.
Miguel
M
Pessanha
Yuri Y
YY
Londer
W Chris
WC
Long
Jill
J
Erickson
P Raj
PR
Pokkuluri
Marianne
M
Schiffer
Carlos A
CA
Salgueiro
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D045287:Desulfuromonas / Q000201:enzymology; D045302:Geobacter / Q000201:enzymology* / Q000235:genetics; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics*; D011522:Protons; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D011522:Protons; C114913:cytochrome C7; D014443:Tyrosine; D006418:Heme; D010649:Phenylalanine