The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a typical hinge peptide. To probe the importance of the structural integrity of the hinge region for efficient intraprotein electron transfer, three mutant enzymes have been constructed: H delta 3 [Sharp, R. E., White, P., Chapman, S. K., & Reid, G. A. (1994) Biochemistry 33, 5115-5120], H delta 6, and H delta 9 in which three, six, and nine amino acids, respectively, were deleted from the hinge region. Intraprotein electron transfer was investigated by steady-state and stopped-flow kinetic analyses. All three hinge-deletion enzymes remained good L-lactate dehydrogenases, as was evident from steady-state experiments with ferricyanide as the electron acceptor and from stopped-flow experiments monitoring flavin reduction. The global effect of these deletions is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of H delta 6 and H delta 9 flavocytochromes b2 is manifested at the first interdomain electron-transfer step (fully reduced FMN-->heme electron transfer), where the rate of heme reduction is the same within experimental error as the steady-state rate of cytochrome c reduction. Thus, interdomain electron transfer is rate limiting in the case of these two hinge-deletion enzymes compared to the wild-type enzyme, where alpha H abstraction from C-2 of L-lactate still contributes substantially to rate limitation. The situation for H delta 3 is more complicated, with more than one interdomain electron-transfer step being affected. Kinetic data, along with the measured deuterium kinetic isotope effects, are discussed in the context of the flavocytochrome b2 catalytic cycle and show that complete structural integrity within the hinge region is essential for efficient interdomain communication.
Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.
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