Lois Geren

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase II. RAPID KINETIC ANALYSIS OF ELECTRON TRANSFER FROM CYTOCHROMEc TO RHODOBACTER SPHAEROIDES CYTOCHROME OXIDASE SURFACE MUTANTS

The reaction between cytochrome c(Cc) and Rhodobacter sphaeroides cytochrome coxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to CuA with an intracomplex rate constant ofk a = 4 × 104 s−1, followed by electron transfer from CuA to heme a with a rate constant of k b = 9 × 104s−1. The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q ≈ D188N/E189Q ≈ wild type, indicating that the acidic residues Asp214, Glu157, Glu148, and Asp195 on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp143, to Phe or Ala decreased the intracomplex electron transfer rate constant k a by 450- and 1200-fold, respectively, without affecting the dissociation constant K D . It therefore appears that the indole ring of Trp143 mediates electron transfer from the heme group of Cc to CuA. These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032–38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem.274, 38051–38060).

The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, has been used to cross-link horse heart cytochrome c to spinach chloroplast plastocyanin. The complex was formed in yields up to 90% and was found to have a stoichiometry of 1 mol plastocyanin per mol cytochrome c. The cytochrome c in the complex was fully reducible by ascorbate and potassium ferrocyanide, and had a redox potential only 25 mV less than that of native cytochrome c. The complex was nearly completely inactive towards succinate-cytochrome c reductase and cytochrome c oxidase, suggesting that the heme crevice region of cytochrome c was blocked. We propose that the carbodiimide promoted the formation of amide cross-links between lysine amino groups surrounding the heme crevice of cytochrome c and complementary carboxyl groups on plastocyanin. It is of interest that the high-affinity site for cytochrome c binding on bovine heart cytochrome c oxidase has recently been found to involve a sequence of subunit II with some homology to the copper-binding sequence of plastocyanin.

Electron transfer between cytochromec and cytochromec peroxidase

The reaction between cytochromec (CC) and cytochromec peroxidase (CcP) is a very attractive system for investigating the fundamental mechanism of biological electron transfer. The resting ferric state of CcP is oxidized by hydrogen peroxide to compound I (CMPI) containing an oxyferryl heme and an indolyl radical cation on Trp-191. CMPI is sequentially reduced to CMPII and then to the resting state CcP by two molecules of CC. In this review we discuss the use of a new ruthenium photoreduction technique and other rapid kinetic techniques to address the following important questions: (1) What is the initial electron acceptor in CMPI? (2) What are the true rates of electron transfer from CC to the radical cation and to the oxyferryl heme? (3) What are the binding domains and pathways for electron transfer from CC to the radical cation and the oxyferryl heme? (4) What is the mechanism for the complete reaction under physiological conditions?

Proton-Dependent Electron Transfer from CuA to Heme a and Altered EPR Spectra in Mutants Close to Heme a of Cytochrome Oxidase

Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g(z) = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. Mutation to aspartate, however, results in an oppositely shifted and split heme a EPR signal of g(z) = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from CuA to heme a. This intrinsic rate is biphasic; the proportion that is slow is pH dependent, as is the relative intensity of the two EPR signal components. At pH 8, the heme a EPR signal at 2.72 is most intense, and the electron transfer rate (CuA to heme a) is 10-130 s(-1), compared to wild-type at 90,000 s(-1). At pH 5.5, the signal at 2.78 is intensified, and a biphasic rate is observed, 50% fast (approximately wild type) and 50% slow (90 s(-1)). The data support the prediction that the hydrogen-bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial CcO. We further demonstrate that the heme a redox potential can be dramatically altered by a nearby carboxyl, whose protonation leads to a proton-coupled electron transfer process.

Chemical modification of interaction between adrenodoxin and cytochrome P450scc

Publisher Summary This chapter discusses the chemical modification of interaction between adrenodoxin and cytochrome P450scc. Cytochrome P450scc is an integral membrane protein located in the inner membrane of adrenal cortex mitochondria that carries out the three-step oxidative side-chain cleavage of cholesterol to form pregnenolone. This chapter presents detailed chemical modification procedures to characterize the interaction between adrenodoxin and cytochrome P450scc. Methods for the specific modification of lysine, arginine, glutamate, and aspartate residues are presented, along with procedures to analyze the effects of these modifications on protein-protein interactions. The limitations and caveats of the chemical modification procedures are discussed. A number of different factors can lead to a change in protein function as a result of the chemical modification of an amino acid residue. For this reason, caution should be used in the interpretation of chemical modification experiments. It is difficult to distinguish between a direct, localized effect on function, and the effect of a global long-range change in conformation accompanying modification.