Francis Millett

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.

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).

Equilibrium binding constants for the group I metal cations with gramicidin-A determined by competition studies and T1+-205 nuclear magnetic resonance spectroscopy

The equilibrium binding constants of the Group I metal cations with gramicidin A in aqueous dispersions of lyso-PC have been determined using a combination of competitive binding with the T1+ ion and T1-205 NMR spectroscopy. The values of the binding constants at 34 degrees C are Li (32.2 M-1), Na (36.9 M-1), K (52.6 M-1), Rb (55.9 M-1), and Cs (54.0 M-1). The equilibrium binding constant for the T1+ ion at this temperature is 582 M-1. The relationships between the binding constants, the free energy of the binding process, and the cation selectivity of the gramicidin A channel are discussed.

Rapid kinetics of membrane potential generation by cytochrome c oxidase with the photoactive Ru(II)‐tris‐bipyridyl derivative of cytochrome c as electron donor

Yeast iso‐1‐cytochrome c covalently modified at cysteine‐102 with (4‐bromomethyl‐4′‐methylbipyridine)[bis(bi‐pyridine)]Ru2+ (Ru‐102‐Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome c oxidase (COX) reconstituted into phospholipid vesicles. Rapid kinetics of membrane potential generation by the enzyme following flash‐induced photoreduction of Ru‐102‐Cyt c heme has been measured and compared to photovoltaic responses observed with Ru(II)(bipy‐ridyl)3 (RuBpy) as the photoreductant [D.L. Zaslavsky et al. (1993) FEBS Lett. 336, 389–393]. At low ionic strength, when Ru‐102‐Cyt c forms a tight electrostatic complex with COX, flash‐activation results in a polyphasic electrogenic response corresponding to transfer of a negative charge to the interior of the vesicles. The initial rapid phase is virtually identical to the 50 μs transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme a by CuA. CuA reduction by Ru‐102‐Cyt c turns out to be not electrogenic in agreement with the peripheral location of visible copper in the enzyme. A millisecond phase (τ ca. 4 ms) following the 50 μs initial part of the response and associated with vectorial translocation of protons linked to oxygen intermediate interconversion in the binuclear centre, can be resolved both with RuBpy and Ru‐102‐Cyt c as electron donors; however, this phase is small in the absence of added H2O2. In addition to these two transients, the flash‐induced electrogenic response in the presence of Ru‐102‐Cyt c reveals a large slow phase of Δψ generation not observed with RuBpy. This phase is completely quenched upon inclusion of 100 μM ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru‐102‐Cyt c 2+ generated by the flash in a solution.

Definition of the Interaction Domain for Cytochrome con the Cytochrome bc 1 Complex STEADY-STATE AND RAPID KINETIC ANALYSIS OF ELECTRON TRANSFER BETWEEN CYTOCHROME c AND RHODOBACTER SPHAEROIDESCYTOCHROME bc 1 SURFACE MUTANTS

The interaction domain for cytochromec on the cytochrome bc 1 complex was studied using a series of Rhodobacter sphaeroidescytochrome bc 1 mutants in which acidic residues on the surface of cytochrome c 1 were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochromebc 1 at low ionic strength resulted in electron transfer from photoreduced heme c to cytochromec 1 with a rate constant ofk et = 6 × 104s−1. Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lowerk et values as well as significantly higher equilibrium dissociation constants and steady-stateK m values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c 1 are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c 1direct the diffusion and binding of cytochrome c from the intramembrane space.

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.

A 19F nuclear magnetic resonance study of the interaction between cytochrome c and cytochrome c peroxidase

The reaction between ferrocytochrome c and yeast cytochrome c peroxidase was studied using cytochrome c derivatives specifically trifluoroacetylated at single lysine amino groups. The only modifications that decreased the reaction rate were those of lysines immediately surrounding the heme crevice, lysines 13, 25, 79, and 87. Modification of lysines 22, 55, 88, and 99 had no effect on the reaction. The 19F chemical shifts of the cytochrome c derivatives trifluoroacetylated at lysines 13, 79, and 87 were not changed upon complex formation with cytochrome c peroxidase, indicating that no detectable conformational changes occurred. The cytochrome c trifluoroacetyl groups had the same T1 values in the paramagnetic fluorocytochrome c peroxidase complex as in the diamagnetic reduced form of the complex, indicating that they were more than 2.3 nm from the paramagnetic iron atom in cytochrome c peroxidase. This is consistent with a separation of at least 1.5-2.0 nm between the iron atom of cytochrome c and the iron atom of cytochrome c peroxidase.

Effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c1 in the cytochrome bc1 complex.

Famoxadone is a new cytochromebc 1 Qo site inhibitor that immobilizes the iron-sulfur protein (ISP) in the bconformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c 1 were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c 1 was found to be 16,000 s−1in bovine cyt bc 1. Binding famoxadone decreased this rate constant to 1,480 s−1, consistent with a decrease in mobility of the ISP. Reverse electron transfer from cytc 1 to 2Fe-2S was found to be biphasic in bovine cyt bc 1 with rate constants of 90,000 and 7,300 s−1. In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s−1. It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc 1 mutants involving residues at the interface between the Rieske protein and cytc 1 and/or cyt b.

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 greater than Lys 25 congruent to Lys 79 approximately equal to Lys 87 greater than Lys 8 approximately equal to Lys 27 approximately equal to Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.

Kinetics of Electron Transfer within Cytochrome bc (1) and Between Cytochrome bc (1) and Cytochrome c.

In this minireview an overview is presented of the kinetics of electron transfer within the cytochrome bc1 complex, as well as from cytochrome bc1 to cytochrome c. The cytochrome bc1 complex (ubiquinone:cytochrome c oxidoreductase) is an integral membrane protein found in the mitochondrial respiratory chain as well as the electron transfer chains of many respiratory and photosynthetic bacteria. Experiments on both mitochondrial and bacterial cyatochrome bc1 have provided detailed kinetic information supporting a Q-cycle mechanism for electron transfer within the complex. On the basis of X-ray crystallographic studies of cytochrome bc1, it has been proposed that the Rieske iron–sulfur protein undergoes large conformational changes as it transports electrons from ubiquinol to cytochrome c1. A new method was developed to study electron transfer within cytochrome bc1 using a binuclear ruthenium complex to rapidly photooxidize cytochrome c1. The rate constant for electron transfer from the iron–sulfur center to cytochrome c1 was found to be 80,000 s−1, and is controlled by the dynamics of conformational changes in the iron–sulfur protein. Moreover, a linkage between the conformation of the ubiquinol binding site and the conformational dynamics of the iron–sulfur protein has been discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method has also been developed to measure electron transfer from cytochrome c1 to cytochrome c. The kinetics of electron transfer are interpreted in light of a new X-ray crystal structure for the complex between cytochrome bc1 and cytochrome c.