Bill Durham

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).

ELECTRON TRANSFER FROM CYTOCHROME B5 TO CYTOCHROME C

The reaction of cytochromeb5 with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments.

Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry of Triacylglycerols and Other Components in Fingermark Samples*

Abstract:  The chemical composition of fingermarks could potentially be important for determining investigative leads, placing individuals at the time of a crime, and has applications as biomarkers of disease. Fingermark samples containing triacylglycerols (TAGs) and other components were analyzed using laser desorption/ionization (LDI) time‐of‐flight mass spectrometry (TOF MS). Only LDI appeared to be useful for this application while conventional matrix‐assisted LDI‐TOF MS was not. Tandem MS was used to identify/confirm selected TAGs. A limited gender comparison, based on a simple t‐distribution and peaks intensities, indicated that two TAGs showed gender specificity at the 95% confidence level and two others at 97.5% confidence. Because gender‐related TAGs differences were most often close to the standard deviation of the measurements, the majority of the TAGs showed no gender specificity. Thus, LDI‐TOF MS is not a reliable indicator of gender based on fingermark analysis. Cosmetic ingredients present in some samples were identified.

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.

A rapid separation technique for overcoming suppression of triacylglycerols by phosphatidylcholine using MALDI-TOF MS

Phospholipids and triacylglycerols (TAGs) are important classes of lipids in biological systems. Rapid methods have been developed for their characterization in crude samples, including MALDI time-of-flight MS. For mixtures, MALDI often selectively shows only some components. For example, phosphatidylcholine (PC) suppresses detection of other lipids. Most rapid MS methods detect either TAGs or phospholipids but not both. Herein, we demonstrate a simple approach to rapidly screen mixtures containing multiple lipid classes. To validate this approach, reference lipids [PC, tripalmitin (PPP), and phosphatidyl-ethanolamine (PE)] and real samples (beef, egg yolk) were used. In a binary mixture with a strong suppressor (PC), PPP was greatly suppressed. After a simple separation, suppression was virtually eliminated. A mixture of nominally nonsuppressing lipids (PE and PPP) was not adversely affected by separation. Ground beef and egg yolk were used to demonstrate detection of known lipid compositions where other methods have missed one or more lipids or lipid classes. Separation was performed using solid phase extraction with a PrepSep florisil column. A 10 min separation allows rapid screening for lipids and changes in lipids. It is sufficient to clearly detect all lipids and overcome suppression effects in complex lipid mixtures.

Electron transfer from cytochromeb5 to cytochromec

The reaction of cytochromeb5 with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments.

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.

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.

Comparison of laser desorption and matrix-assisted laser desorption/ionization for ruthenium and osmium trisbipyridine complexes using Fourier transform mass spectrometry.

Metal-bipyridine complexes are a vehicle for developing approaches for studying the fluorescence of gas-phase ions; however, conclusions regarding fluorescence behavior depend on explicitly identifying the ionic species in the gas phase. [Ru(bpy)3]X2 and [Os(bpy3)]X2, (where bpy=2,2′-bipyridine and X=Cl or PF6), were studied using direct laser desorption (LD) and matrix-assisted laser desorption/ionization (MALDI) using Fourier transform mass spectrometry (FTMS). LD spectra of the PF6 salt of the Ru and Os complexes reveal counterion attachment, fluoride transfer, and significant losses of H for a number of peaks. LD of the chloride salt complexes produced loss of a single bpy ligand, chloride attachment, and losses of H. Spectra of [Ru(bpy3)]X2 where X=BF4−, CF3SO3−, and SCN− were also collected using LD and compared with the spectra for Cl2 and PF6 salts. Regardless of counterion, loss of H is observed in LD spectra. MALDI spectra of the trisbipyridyl complexes using 2,5-dihydroxybenzoic acid (DHB) and sinapinic acid (SA) as the matrix were also obtained. The spectra using SA as matrix show intact molecular ion peaks with very little fragmentation and no counterion attachment. Unlike SA, the spectra obtained using DHB look similar to LD spectra with significant losses of H. Our results are consistent with a reaction scheme for hydrogen loss from a carbon that also involves breaking of the metal-nitrogen bond, rotation of a pyridine ring, and re-formation of an ortho-metallated complex by a metal-C bond. These results demonstrate the importance of ion generation method and the utilization of FTMS for correct characterization of metal poly(pyridyl) complexes.

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?