Isolation and characterization of cytochrome c550 from the methylamine-oxidizing electron-transport chain of Thiobacillus versutus.
The isolation and purification of cytochrome c550 from the methylamine-oxidizing electron-transport chain in Thiobacillus versutus is reported. The cytochrome is a single-heme-containing type I cytochrome c with a relative molecular mass of 16 +/- 1 kDa, an isoelectric point of 4.6 +/- 0.1, a midpoint potential of 272 +/- 3 mV at pH less than 4 and 255 +/- 5 mV at pH = 7.0, and an axial coordination of the Fe by a methionine and a histidine. The midpoint potential decreases with increasing pH due to the deprotonation of a group tentatively identified as a propionate (pKa = 6.5 +/- 0.1 and 6.7 +/- 0.1 in the oxidized and reduced protein, respectively) and a change in the Fe coordination at pH greater than 10. The electron-self-exchange rate appears to depend strongly on the ionic strength of the solution and is relatively insensitive to changes in pH. At 313 K and pH 5.2 the electron-exchange rate amounts to 0.7 x 10(2) M-1 s-1 and 5.3 x 10(2) M-1 s-1 at I = 40 mM and I = 200 mM, respectively. Amino acid composition and molar absorption coefficients at various wavelengths are reported. Resonances of heme protons and the epsilon H3 group of the ligand methionine of the Fe have been identified in the 1H-NMR spectrum of the reduced as well as the oxidized cytochrome.
Lommen
A
A
Gorlaeus Laboratories, Leiden University, The Netherlands.
Ratsma
A
A
Bijlsma
N
N
Canters
G W
GW
van Wielink
J E
JE
Frank
J
J
van Beeumen
J
J
D000596:Amino Acids / Q000032:analysis; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification*; D004579:Electron Transport; D006418:Heme / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008744:Methylamines / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry; D013696:Temperature; D013855:Thiobacillus / Q000201:enzymology* / Q000378:metabolism
D000596:Amino Acids; D003574:Cytochrome c Group; D008744:Methylamines; D006418:Heme; C031732:cytochrome C-550