In vitro kinetics of reduction of cytochrome c554 isolated from the reaction center of the green phototrophic bacterium, Chloroflexus aurantiacus.
The photochemical reaction center in the green bacterium Chloroflexus aurantiacus is similar to that found in purple phototrophic bacteria and interacts with a multiheme membrane-bound cytochrome. We have examined the kinetics of reduction of the pure solubilized reaction center cytochrome by laser flash photolysis of solutions containing lumiflavin or FMN. Reduction by lumiflavin semiquinone followed single exponential kinetics and the observed rate constant (kobs) was linearly dependent on protein concentration (k = 1.8 X 10(7) M-1s-1 heme-1). This result suggests either that the four hemes have similar reduction rate constants which cannot be resolved or that there are large differences in rate constant and only the most reactive heme (or hemes) was observed under these conditions. To determine the relative reactivities of the four hemes, we varied the extent of heme reduction at a single total protein concentration. As the hemes were progressively reduced by steady-state illumination prior to laser flash photolysis, kobs for the reaction with fully reduced lumiflavin decreased nonlinearly. Second-order rate constants for the four hemes were assigned by nonlinear least-squares analysis of kobs vs oxidized heme concentration data. The second-order rate constants obtained in this way for the highest and lowest potential hemes differed by a factor of about 20, which is larger than expected for c-type cytochromes based on redox potential alone (a factor of about 3 would be expected). This is interpreted as being due to differences in steric accessibility. Relative to the highest potential heme, which is as reactive as a typical c-type cytochrome, we estimated a steric effect of approximately twofold for heme 2, and steric effects of approximately fivefold for hemes 3 and 4. Using fully reduced FMN as reductant of oxidized cytochrome, ionic strength effects indicate a minus-minus interaction, with approximately a -2 charge near the site of reduction of the highest potential heme.
Meyer
T E
TE
Department of Biochemistry, University of Arizona, Tucson 85721.
Tollin
G
G
Cusanovich
M A
MA
Freeman
J C
JC
Blankenship
R E
RE
D001419:Bacteria / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis*; D003574:Cytochrome c Group / Q000302:isolation & purification / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010782:Photolysis; D045322:Photosynthetic Reaction Center Complex Proteins
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D005415:Flavins; D045322:Photosynthetic Reaction Center Complex Proteins; C083951:cytochrome c553; C001922:lumiflavin; D006418:Heme; D005486:Flavin Mononucleotide; C022302:cytochrome C-552