Definition of the Interaction Domain and Electron Transfer Route between Cytochrome c and Cytochrome Oxidase.

The reaction between cytochrome c (Cc) and cytochrome c oxidase (CcO) was studied using horse cytochrome c derivatives labeled with ruthenium trisbipyridine at Cys 39 (Ru-39-Cc). Flash photolysis of a 1:1 complex between Ru-39-Cc and bovine CcO at a low ionic strength resulted in the electron transfer from photoreduced heme c to CuA with an intracomplex rate constant of k3 = 6 × 104 s-1. The K13A, K72A, K86A, and K87A Ru-39-Cc mutants had nearly the same k3 value but bound much more weakly to bovine CcO than wild-type Ru-39-Cc, indicating that lysines 13, 72, 86, and 87 were involved in electrostatic binding to CcO, but were not involved in the electron transfer pathway. The Rhodobacter sphaeroides (Rs) W143F mutant (bovine W104) caused a 450-fold decrease in k3 but did not affect the binding strength with CcO or the redox potential of CuA. These results are consistent with a computational model for Cc-CcO (Roberts and Pique ( 1999 ) J. Biol. Chem. 274 , 38051 - 38060 ) with the following electron transfer pathway: heme c → CcO-W104 → CcO-M207 → CuA. A crystal structure for the Cc-CcO complex with the proposed electron transfer pathway heme c → Cc-C14 → Cc-K13 → CcO-Y105 → CcO-M207 → CuA ( S. Shimada ( 2017 ) EMBO J. 36 , 291 - 300 ) is not consistent with the kinetic results because the K13A mutation had no effect on k3. Addition of 40% ethylene glycol (as present during the crystal preparation) decreased k3 significantly, indicating that it affected the conformation of the complex. This may explain the discrepancy between the current results and the crystallographic structure.

Proteo-lipobeads for the oriented encapsulation of membrane proteins.

As a surrogate of live cells, proteo-lipobeads are presented, encapsulating functional membrane proteins in a strict orientation into a lipid bilayer. Assays can be performed just as on live cells, for example using fluorescence measurements. As a proof of concept, we have demonstrated proton transport through cytochrome c oxidase.

Design and use of photoactive ruthenium complexes to study electron transfer within cytochrome bc1 and from cytochrome bc1 to cytochrome c.

The cytochrome bc1 complex (ubiquinone:cytochrome c oxidoreductase) is the central integral membrane protein in the mitochondrial respiratory chain as well as the electron-transfer chains of many respiratory and photosynthetic prokaryotes. Based on X-ray crystallographic studies of cytochrome bc1, a mechanism has been proposed in which the extrinsic domain of the iron-sulfur protein first binds to cytochrome b where it accepts an electron from ubiquinol in the Qo site, and then rotates by 57° to a position close to cytochrome c1 where it transfers an electron to cytochrome c1. This review describes the development of a ruthenium photooxidation technique to measure key electron transfer steps in cytochrome bc1, including rapid electron transfer from the iron-sulfur protein to cytochrome c1. It was discovered that this reaction is rate-limited by the rotational dynamics of the iron-sulfur protein rather than true electron transfer. A conformational linkage between the occupant of the Qo ubiquinol binding site and the rotational dynamics of the iron-sulfur protein was discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method is also described for the measurement of electron transfer from cytochrome c1 to cytochrome c. This article is part of a Special Issue entitled: Respiratory Complex III and related bc complexes.

Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase.

This review describes the development and application of photoactive ruthenium complexes to study electron transfer and proton pumping reactions in cytochrome c oxidase (CcO). CcO uses four electrons from Cc to reduce O(2) to two waters, and pumps four protons across the membrane. The electron transfer reactions in cytochrome oxidase are very rapid, and cannot be resolved by stopped-flow mixing techniques. Methods have been developed to covalently attach a photoactive tris(bipyridine)ruthenium group [Ru(II)] to Cc to form Ru-39-Cc. Photoexcitation of Ru(II) to the excited state Ru(II*), a strong reductant, leads to rapid electron transfer to the ferric heme group in Cc, followed by electron transfer to Cu(A) in CcO with a rate constant of 60,000s(-1). Ruthenium kinetics and mutagenesis studies have been used to define the domain for the interaction between Cc and CcO. New ruthenium dimers have also been developed to rapidly inject electrons into Cu(A) of CcO with yields as high as 60%, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism.

Photoinduced electron transfer in cytochrome bc1: Dynamics of rotation of the Iron-sulfur protein during bifurcated electron transfer from ubiquinol to cytochrome c1 and cytochrome bL.

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc1 (cyt bc1) were studied using a binuclear ruthenium complex to rapidly photooxidize cyt c1. When cyt c1, the iron­sulfur center Fe2S2, and cyt bH were reduced before the reaction, photooxidation of cyt c1 led to electron transfer from Fe2S2 to cyt c1 with a rate constant of ka = 80,000 s-1, followed by bifurcated reduction of both Fe2S2 and cyt bL by QH2 in the Qo site with a rate constant of k2 = 3000 s-1. The resulting Q then traveled from the Qo site to the Qi site and oxidized one equivalent each of cyt bL and cyt bH with a rate constant of k3 = 340 s-1. The rate constant ka was decreased in a nonlinear fashion by a factor of 53 as the viscosity was increased to 13.7. A mechanism that is consistent with the effect of viscosity involves rotational diffusion of the iron­sulfur protein from the b state with reduced Fe2S2 close to cyt bL to one or more intermediate states, followed by rotation to the final c1 state with Fe2S2 close to cyt c1, and rapid electron transfer to cyt c1.

The acidic domain of cytochrome c1 in paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc1 complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c(552).

The cytochrome bc(1) complex is a key component in several respiratory pathways. One of the characteristics of the eukaryotic complex is the presence of a small acidic subunit, which is thought to guide the interaction of the complex with its electron acceptor and facilitate electron transfer. Paracoccus denitrificans represents the only example of a prokaryotic organism in which a highly acidic domain is covalently fused to the cytochrome c(1) subunit. In this work, a deletion variant lacking this acidic domain has been produced and purified by affinity chromatography. The complex is fully intact as shown by its X-ray structure, and is a dimer (Kleinschroth et al., subm.) compared to the tetrameric (dimer-of-dimer) state of the wild-type. The variant complex is studied by steady-state kinetics and flash photolysis, showing wild type turnover and a virtually identical interaction with its substrate cytochrome c(552).

Accelerated Evolution of Cytochrome c in Higher Primates, and Regulation of the Reaction between Cytochrome c and Cytochrome Oxidase by Phosphorylation.

Cytochrome c (Cc) underwent accelerated evolution from the stem of the anthropoid primates to humans. Of the 11 amino acid changes that occurred from horse Cc to human Cc, five were at Cc residues near the binding site of the Cc:CcO complex. Single-point mutants of horse and human Cc were made at each of these positions. The Cc:CcO dissociation constant KD of the horse mutants decreased in the order: T89E > native horse Cc > V11I Cc > Q12M > D50A > A83V > native human. The largest effect was observed for the mutants at residue 50, where the horse Cc D50A mutant decreased KD from 28.4 to 11.8 µM, and the human Cc A50D increased KD from 4.7 to 15.7 µM. To investigate the role of Cc phosphorylation in regulating the reaction with CcO, phosphomimetic human Cc mutants were prepared. The Cc T28E, S47E, and Y48E mutants increased the dissociation rate constant kd, decreased the formation rate constant kf, and increased the equilibrium dissociation constant KD of the Cc:CcO complex. These studies indicate that phosphorylation of these residues plays an important role in regulating mitochondrial electron transport and membrane potential ΔΨ.

Photoinitiated electron transfer within the Paracoccus denitrificans cytochrome bc1 complex: mobility of the iron-sulfur protein is modulated by the occupant of the Q(o) site.

Domain rotation of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c(1) redox centers plays a key role in the mechanism of the cyt bc(1) complex. Electron transfer within the cyt bc(1) complex of Paracoccus denitrificans was studied using a ruthenium dimer to rapidly photo-oxidize cyt c(1) within 1 µs and initiate the reaction. In the absence of any added quinol or inhibitor of the bc(1) complex at pH 8.0, electron transfer from reduced ISP to cyt c(1) was biphasic with rate constants of k(1f) = 6300 ± 3000 s(-1)and k(1s) = 640 ± 300 s(-1) and amplitudes of 10 ± 3% and 16 ± 4% of the total amount of cyt c(1) photooxidized. Upon addition of any of the P(m) type inhibitors MOA-stilbene, myxothiazol, or azoxystrobin to cyt bc(1) in the absence of quinol, the total amplitude increased 2-fold, consistent with a decrease in redox potential of the ISP. In addition, the relative amplitude of the fast phase increased significantly, consistent with a change in the dynamics of the ISP domain rotation. In contrast, addition of the P(f) type inhibitors JG-144 and famoxadone decreased the rate constant k(1f) by 5-10-fold and increased the amplitude over 2-fold. Addition of quinol substrate in the absence of inhibitors led to a 2-fold increase in the amplitude of the k(1f) phase. The effect of QH(2) on the kinetics of electron transfer from reduced ISP to cyt c(1) was thus similar to that of the P(m) inhibitors and very different from that of the P(f) inhibitors. The current results indicate that the species occupying the Q(o) site has a significant conformational influence on the dynamics of the ISP domain rotation.

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.

Probing the Paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.

Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1CF) and cyt c(552F). A new ruthenium cyt c(552F) derivative labeled at C23 (Ru(z)-23-c(552F)) was designed to measure rapid electron transfer with cyt c(1CF) in the physiological direction using flash photolysis. The bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected at an ionic strength of <35 mM with the ruthenium photoexcitation technique in which k(12) became very rapid (3 x 10(9) M(-1) s(-1)) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ru(z)-23-c(552F) and c(1CF) was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was >30 microM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ru(z)-39-Cc formed a tight 1:1 complex with cyt c(1CF) at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s(-1). A group of cyt c(1CF) variants in the presumed docking site were generated on the basis of information from the yeast cyt bc(1)-cyt c cocrystal structure. Kinetic analysis of cyt c(1CF) mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c(552F) and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions.

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.

Single-electron photoreduction of the P(M) intermediate of cytochrome c oxidase.

The P(M)-->F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P(M) state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is approximately 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F-->O transition, but is significantly slower than formation of the F state from the P(R) intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70-90 micros). The approximately 0.3 ms P(M)-->F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P(M)-->F transition is generated much later, with a time constant of 1.3 ms. In addition, the P(M)-->F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with tau values of 0.18 and 0.85 ms.

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)]X(2) and [Os(bpy(3))]X(2), (where bpy = 2,2'-bipyridine and X = Cl or PF(6)), were studied using direct laser desorption (LD) and matrix-assisted laser desorption/ionization (MALDI) using Fourier transform mass spectrometry (FTMS). LD spectra of the PF(6) 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(bpy(3)]X(2) where X = BF(4)(-), CF(3)SO(3)(-), and SCN(-) were also collected using LD and compared with the spectra for Cl(2) and PF(6) 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 metalz.sbnd;nitrogen bond, rotation of a pyridine ring, and re-formation of an ortho-metallated complex by a metalz.sbnd;C bond. These results demonstrate the importance of ion generation method and the utilization of FTMS for correct characterization of metal poly(pyridyl) complexes.

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 bc (1) complex, as well as from cytochrome bc (1) to cytochrome c. The cytochrome bc (1) 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 bc (1) 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 bc (1), it has been proposed that the Rieske iron-sulfur protein undergoes large conformational changes as it transports electrons from ubiquinol to cytochrome c (1). A new method was developed to study electron transfer within cytochrome bc (1) using a binuclear ruthenium complex to rapidly photooxidize cytochrome c (1). The rate constant for electron transfer from the iron-sulfur center to cytochrome c (1) 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 c (1) to cytochrome c. The kinetics of electron transfer are interpreted in light of a new X-ray crystal structure for the complex between cytochrome bc (1) and cytochrome c.

The 1997 North American Interagency Intercomparison of Ultraviolet Spectroradiometers Including Narrowband Filter Radiometers.

The fourth North American Intercomparison of Ultraviolet Monitoring Spectroradiometers was held September 15 to 25, 1997 at Table Mountain outside of Boulder, Colorado, USA. Concern over stratospheric ozone depletion has prompted several government agencies in North America to establish networks of spectroradiometers for monitoring solar ultraviolet irradiance at the surface of the Earth. The main purpose of the Intercomparison was to assess the ability of spectroradiometers to accurately measure solar ultraviolet irradiance, and to compare the results between instruments of different monitoring networks. This Intercomparison was coordinated by NIST and NOAA, and included participants from the ASRC, EPA, NIST, NSF, SERC, USDA, and YES. The UV measuring instruments included scanning spectroradiometers, spectrographs, narrow band multi-filter radiometers, and broadband radiometers. Instruments were characterized for wavelength accuracy, bandwidth, stray-light rejection, and spectral irradiance responsivity. The spectral irradiance responsivity was determined two to three times outdoors to assess temporal stability. Synchronized spectral scans of the solar irradiance were performed over several days. Using the spectral irradiance responsivities determined with the NIST traceable standard lamp, and a simple convolution technique with a Gaussian slit-scattering function to account for the different bandwidths of the instruments, the measured solar irradiance from the spectroradiometers excluding the filter radiometers at 16.5 h UTC had a relative standard deviation of ±4 % for wavelengths greater than 305 nm. The relative standard deviation for the solar irradiance at 16.5 h UTC including the filter radiometer was ±4 % for filter functions above 300 nm.

Gas chromatography-mass spectrometry of JWH-018 metabolites in urine samples with direct comparison to analytical standards.

JWH-018 (1-pentyl-3-(1-naphthoyl)indole) is one of numerous potential aminoalkylindoles contained in products marketed as 'K2' or 'Spice'. Investigation of the urinary metabolites from consumption of these compounds is important because they are banned in the United States and many European countries. An efficient extraction procedure and gas chromatography-mass spectrometry (GC-MS) method were developed for detection of 'K2' metabolites in urine from individuals suspected of using these products. Analytical standards were used to elucidate the structure-specific mass spectral fragmentations and retention properties to confirm proposed identifications and support quantitative studies. A procedure for the synthesis of one of these metabolites (5-hydroxypentyl JWH-018) was also developed. Results are comparable to existing LC-MS/MS methods, with the same primary metabolites detected. The specific metabolite hydrolysis products include 4-hydroxpentyl, 5-hydroxypentyl, and N-pentanoic acid derivatives.

Laser desorption/ionization time-of-flight mass spectrometry of triacylglycerols and other components in fingermark samples.

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.

Chapter 5 Use of ruthenium photooxidation techniques to study electron transfer in the cytochrome bc1 complex.

Ruthenium photooxidation methods are presented to study electron transfer between the cytochrome bc(1) complex and cytochrome c and within the cytochrome bc(1) complex. Methods are described to prepare a ruthenium cytochrome c derivative, Ru(z)-39-Cc, by labeling the single sulfhydryl on yeast H39C;C102T iso-1-Cc with the reagent Ru(bpz)(2)(4-bromomethyl-4'-methylbipyridine). The ruthenium complex attached to Cys-39 on the opposite side of Cc from the heme crevice does not affect the interaction with cyt bc(1). Laser excitation of reduced Ru(z)-39-Cc results in photooxidation of heme c within 1 microsec with a yield of 20%. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc(1) and Ru(z)-39-Cc leads to electron transfer from heme c(1) to heme c with a rate constant of 1.4 x 10(4) s(-1). Methods are described for the use of the ruthenium dimer, Ru(2)D, to photooxidize cyt c(1) in the cytochrome bc(1) complex within 1 microsec with a yield of 20%. Electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c(1) was detected with a rate constant of 6 x 10(4) s(-1) in R. sphaeroides cyt bc(1) with this method. This electron transfer step is rate-limited by the rotation of the Rieske iron-sulfur protein in a conformational gating mechanism. This method provides critical information on the dynamics of rotation of the iron-sulfur protein (ISP) as it transfers electrons from QH(2) in the Q(o) site to cyt c(1). These ruthenium photooxidation methods can be used to measure many of the electron transfer reactions in cytochrome bc(1) complexes from any source.

Chapter 28 Use of ruthenium photoreduction techniques to study electron transfer in cytochrome oxidase.

Ruthenium photoreduction methods are described to study electron transfer from cytochrome c to cytochrome c oxidase and within cytochrome oxidase. Methods are described to prepare a ruthenium cytochrome c derivative Ru-39-Cc, by labeling the single sulfhydryl group on horse K39C with (4-bromomethyl-4'methylbipyridine) (bis-bipyridine)ruthenium(II). The ruthenium complex attached to Cys-39 on the opposite side of the heme crevice does not interfere with the interaction with cytochrome oxidase. Laser flash photolysis of a 1:1 complex between Ru-39-Cc and bovine cytochrome oxidase results in photoreduction of heme c within 1 microsec, followed by electron transfer from heme c to Cu(A) in cytochrome oxidase with a rate constant of 60,000 s(-1) and from Cu(A) to heme a with a rate constant of 20,000 s(-1). A new ruthenium dimer, Ru(2)Z, has been developed to reduce Cu(A) within 1 microsec with a yield of 60%, followed by electron transfer from Cu(A) to heme a and then to the heme a(3)/Cu(B) binuclear center. Methods are described to measure the single-electron reduction of each of the intermediates involved in reduction of oxygen to water by cytochrome oxidase, including P(m), F, O(H), and E.

Reducing fragmentation observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of triacylglycerols in vegetable oils.

Edible oils consist primarily of triacylglycerols (TAGs). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of the oils are typically dominated by sodium adducts of these TAGs but also show prominent fragment ions (that do not contain sodium), which can interfere with analytical measurements of other components in oils. The fragments seemingly correspond to the loss of a fatty acid moiety from the sodiated TAGs as a sodium salt: RCOONa. However, a previous study suggested that the fragments actually arise from nearly complete fragmentation of unseen protonated TAGs. These authors suggested that the fragmentation occurs so rapidly and completely that protonated TAGs are not normally observed in the spectra of these oils. In this paper, we present evidence to support their theory and also demonstrate an approach to eliminate these interfering ions from the MALDI-TOF mass spectra via addition of a base to the matrix/sample mixture. The added base does not impede formation of the sodiated TAGs, but does significantly reduce the amount of fragments observed. We propose that this occurs by depleting the H+ ions from the matrix, thus preventing the formation of significant numbers of protonated TAGs in the first place. For measurements by MALDI-TOF, the relative abundances of the fragment ions are related to the strength of the base, and can be almost completely eliminated. However, in longer time-scale experiments such as in post-source decay and Fourier transform mass spectrometry, sodiated and non-sodiated diacylglycerol (DAG)-like fragments are present in spectra, regardless of whether or not a base is added to the sample.