Electron transfer and spectral alpha-band properties of the di-heme protein cytochrome c4 from Pseudomonas stutzeri.
Cytochrome c4 is a 190-residue protein active in the aerobic and anaerobic respiration of several bacteria. We have isolated Pseudomonas stutzeri (ATCC no. 11607) cytochrome c4 by an optimized growth procedure following factorial design. The ultraviolet/visible spectra of reduced cytochrome c4 have a composite alpha/beta band which can be resolved into six components. One of these seems to be specific for the high-potential heme group. The kinetics for full oxidation and reduction with the two inorganic redox couples, [Co(terpy)2]2+/3+ and [Co(bipy)3]2+/3+, is formally compatible with either bi- or tri-exponential kinetics. The former would be in line with weak interaction between the heme groups, the latter with notable interaction effects. Arguments in favour of the latter and a cooperative two-electron transfer pattern are given. All phases are approximately proportional to the Co-complex concentration, implying that intramolecular electron transfer in this time range is unlikely. The rate constants are in the range (0.7-80) x 10(4) M-1 s-1 at pH = 7.6 (Tris) and 0.1 M NaCl and very little dependent on the ionic strength in the range 0.1-0.3 M. The reduction potentials could be calculated from the forward and reverse rate constant ratios. The values are 241 +/- 5 and 328 +/- 2 mV (Nernst hydrogen electrode) if bi-exponential kinetics is used and interaction between the heme groups disregarded. The intrinsic microscopic reduction potential values are closer when the tri-exponential, cooperative model is used as this model transfers 30-40 mV to electrostatically dominated interaction potentials. The overall electron transfer pattern can be related to the recently determined crystal structure of the P. stutzeri cytochrome c4.
Conrad
L S
LS
Chemistry Department A, Technical University of Denmark, Lyngby.
Karlsson
J J
JJ
Ulstrup
J
J
D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006418:Heme / Q000032:analysis*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D003574:Cytochrome c Group; C034186:cytochrome C4; D006418:Heme