Abstract
The interaction of the Rhodospirillum rubrum cytochrome bc1 complex with R. rubrum cytochrome c2 and horse cytochrome c was studied using specific lysine modification and ionic strength dependence methods. In order to define the reaction domain on cytochrome c2, several fractions consisting of mixtures of singly labeled carboxydintrophenyl-cytochrome c2 derivatives were employed. Fraction A consisted of a mixture of derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2, while fractions C1, C2, C3, and C4 were mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The rate of the reaction of fraction A was found to be nearly the same as that of native cytochrome c2. However, the rate constants of fractions C1-C4 were found to be more than 20-fold smaller than that of native cytochrome c2. These results indicate that lysine residues surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. Since the same domain is involved in the reaction with the photosynthetic reaction center, cytochrome c2 must undergo some type of rotational or translational diffusion during electron transport in R. rubrum. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse cytochrome c also involves the heme crevice domain.
Highlights
Joan Hall$, AidasKriaucionasQ,David KnaffQT,and FrancisMillettST From the $Department of Chemistry and Biochemistry, UniuersityofArkansas, Fayetteuille, Arkansas 72701and the §Departmentof Chemistry and Biochemistry, Texas Tech Uniuersity, Lubbock, Texas 79409
The interactionof the Rhodospirillum rubrum cyto- Electrontransferthroughthe cytochrome bc, complex is chrome b e l complex with R. rubrum cytochromec2 and accompanied by vectorial protontranslocationacrossthe horse cytochrome c was studied using specific lysine membrane, which is coupled to ATP synthesis.The cytomodification and ionic strength dependence methods. chrome bel complex has been purified from Rhodospirillum
The results reported here demonstrate the importance of electrostatic interactions in the reaction of the R. rubrum cytochrome bcl complex with its natural oxidantcytochrome c2,as well as with horse cytochrome c
Summary
Thepurplephotosyntheticbacteria havebeen found to on the front side of R. rubrum cytochrome c2 with 4-chlorocontain a cytochrome bc, complex that is similar in many respects to the mitochondriaclytochrome bcl complex (Crofts and Wraight, 1983; Hauska et al, 1983). The cytochromebc, complex transfers electrons from ubiquinol to cytochrome cp, which in turn reduces the. Since the same domain oncytochrome c2is involved in the reaction with the reaction center (Hallet al., 1987), cytochromec2must undergo some type of rotational or translational diffusion during electron transport from the cytochrome bcl complex to thereaction center. The synthesis of reduced ubiquinol, QoCloBr(Hz),h' as been reported in the literature (Yu and Yu, 1982).The 4-carboxy-2,6-dinitrophenyl (CDNP) cytochrome c2 derivatives were prepared as described by. Trifluoroacetyl (CF,CO-) derivatives of horse cytochrome c were prepared by the procedures of Smith etal. All other chemicals were purchased from Fisher and were reagent grade
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