Radical scavenging and electron-transfer reactions in Polyporus Versicolor laccase: A pulse radiolysis study

The interaction of the radicals OH^?, t-BuO^?, e_aq^?, CO₂^XXX and O₂^XXX with the copper oxidase. laccase. from Polyporus, has been studied by the pulse-radiolysis technique. Each of these radicals formed transient adducts with a broad absorption maximum around 310 nm. Analysis of the optical properties and of the very fast rates of formation of these compounds shows that each radical interacts with a limited number of sites on the polypeplide part of the protein amongst R-S-S-R. histidine and aromatic residues. Interaction with the carbonyl group of some of the peptide bonds is also possible. The few target sites are probably hit simultaneously and electron transfer between these sites may also occur. In all cases, in a subsequent step, intramolecular electron transfer from the polypeptide radical adducts leads to a partial reduction of the blue type-1 Cu²⁺ with rates varying between 10³ and 10⁴ s^?1. Further reduction of the type-1 Cu²⁺ occurs through a slow intermolecular reaction between two laccase radical transient adducts. In the case of CO^XXX₂ and O^XXX₂, this slow reduction could alternatively be due to an intermolecular reaction between laccase and CO^XXX₂ or O^XXX₂. The oxidant radicals OH^?. Br^XXX₂ and (SCN)^XXX₂, which formed radical adducts with fully ascorbate-reduced laccase, did not induce any type-1 copper reoxidation.     

Heme-peptide/protein ions and phosphorous ligands: search for site-specific addition reactions

High-resolution Fourier transform ion cyclotron resonance mass spectrometry is employed to gain thorough kinetics and thermodynamics information on the reaction of free and ligated heme-type ions with selected ligands, with the aim of obtaining an insight into the coordination environment of the prosthetic group in a variety of biomolecular ions. Adopting a stepwise approach towards systems of increasing complexity, we examined the reactivity of free gaseous iron(III) protoporphyrin IX ions, Fe(III)-heme(+), of the charged species from microperoxidase-11 (MP11) (covalently peptide bound heme), and of the multiply charged ions from heme proteins, namely, cytochrome c (cyt c) and myoglobin (examples of noncovalently protein bound hemes). Among an array of test compounds allowed to react with Fe(III)-heme(+), OP(OMe)(3) and P(OMe)(3) proved to be similarly efficient ligands in the first addition step, yet displayed markedly distinct reactivity towards heme iron already engaged in axial coordination. The ease with which P(OMe)(3) acts as a second axial ligand is exploited to probe structural and conformational features of biomolecular ions. In this way, circumstantial evidence is gained of a folded conformation of +2 charge state ions from MP11 and an elongated one for the +3 charge state ions. Similarly, both the general reaction pattern and detailed kinetics and thermodynamics data point to a regiospecific addition reaction of P(OMe)(3) directed at the heme iron within multiply charged ions from cyt c. This unprecedented example of ion-molecule reaction which specifically involves a prosthetic group belonging to protein ions stands in contrast to the multiple, nonspecific interactions established by OP(OMe)(3) molecules with the protonated sites of multiply charged cyt c and apomyoglobin ions. This finding may develop and provide sensitive probes of the structure and bonding features of protein ions in the gas phase.     

Theory of the Stark Effect in protein systems containing an electron donor–acceptor couple

Mixed-valency can occur in a variety of biological systems, such as the Cu(I)–Cu(II) pair in hemocyanin, Fe(II)–Fe(III) in many iron–oxo and iron–sulfur proteins, and Mn(II)–Mn(III) or Mn(III)–Mn(IV) in the photosynthetic water oxidase. The characterization of the ground states of such systems often has been controversial. Stark Effect spectroscopy is proving to be a valuable tool for the elucidation of systems of this type. The purpose of the present work is to develop theory for the spectral lineshape for the case where the electron donor and acceptor are coupled directly in a strong electric field. A mixed-valence dimer with an applied electric field aligned along the internuclear axis is studied using a two-site small-polaron model. Potential energy surfaces are calculated in the adiabatic (Born–Oppenheimer) approximation. It is shown that two nuclear coordinates (one totally symmetric and one antisymmetric) are coupled to the electronic motion, whereas only the antisymmetric coordinate is coupled in the absence of an electric field. For a strongly localized system, such as a protein system where electron donor and acceptor sites are separated by large distances, the potential surfaces become highly asymmetrical, but coupling to the totally symmetric mode is not significant. For a localized case corresponding to a valence-trapped two-metal cluster, the displacement along the totally symmetric coordinate is directly proportional to the applied field strength. Along the antisymmetric coordinate, the lowest potential surface is an asymmetric double well. For a delocalized (valence-averaged) two-metal cluster, there is significant displacement along the antisymmetric coordinate, an effect which also vanishes in the absence of an applied field. Contributions to the linewidth are estimated. Localized systems show larger field-induced shift in frequency maximum, whereas delocalized systems show greater field-induced line broadening.     

Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe

The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1^fd, which is cleaved off after mitochondrial import. The physiological function of Etp1^fd is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1^fd has a melting temperature more than 20 °C higher than adrenodoxin. The three-dimensional structure of Etp1^fd has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1^fd with adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1^fd and adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.     

Phosphotransfer reactions as a means of G protein activation

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound subunits and free dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTR The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews). (Mol Cell Biochem 157: 593, 1996)Recipient of Servier Investigator Award     

The kinetics and mechanisms of the reactions of iron(III) with quercetin and morin

The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with the flavonoids quercetin and morin have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M. Mechanisms have been proposed which account satisfactorily for the kinetic data. The data are consistent with a mechanism in which the metal:ligand complex formed initially on reaction of iron(III) with the ligand subsequently decomposes through an electron transfer step. Morin forms a 1:1 metal:ligand complex while quercetin forms a 2:1 metal:ligand complex. Both ligands showed evidence for the involvement of the iron hydroxo dimer Fe2(OH)2(4+) in the complex formation reaction at the hydroxy-carbonyl moiety. The iron(III) assisted decomposition of the initial iron(III) complex formed was also investigated and the rate constants evaluated. Both the complex formation and subsequent electron transfer reactions of iron(III) with these ligands were monitored using UV-visible spectrophotometry. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time dependant spectra.     

Present status and future directions of research in electron-transfer mediated by DNA

 This commentary article presents an overview of recent experimental results on DNA-mediated electron transfer (ET) from the perspective of semiclassical ET theory. The question concerning whether or not DNA can act as a wire is addressed. Much of the article focuses on a discussion of the decay of electronic coupling (β) between electron donors and acceptors with increasing donor/acceptor separation in DNA and in protein systems. In particular, the dependence of the electronic coupling itself (H _AB) on the energy gap between the tunneling energy of the reactants and the virtual ionic states of the DNA bridge is highlighted. The article concludes by suggesting that future experimental and theoretical work in this field should focus on the tunneling gap energies of the systems studied and that special attention should be paid to systems that are likely to be in the "small tunneling gap" regime. It is these systems that are expected to exhibit enhanced electronic couplings and consequently enhanced rates of long-distance ET. Received, accepted: 5 January 1998     

Nonequilibrium,multiple-timescale simulations of ligand-receptor interactions in structured protein systems

Predicting the long-time, nonequilibrium dynamics of receptor-ligand interactions for structured proteins in a host fluid is a formidable task, but of great importance to predicting and analyzing cell-signaling processes and small molecule drug efficacies. Such processes take place on timescales on the order of milliseconds to seconds, so "brute-force" real-time, molecular or atomic simulations to determine absolute ligand-binding rates to receptor targets and over a statistical ensemble of systems are not currently feasible. In the current study, we implement on real protein systems a previously developed 3-5 hybrid molecular dynamics/Brownian dynamics algorithm, which takes advantage of the underlying, disparate timescales involved and overcomes the limitations of brute-force approaches. The algorithm is based on a multiple timescale analysis of the total system Hamiltonian, including all atomic and molecular structure information for the system: water, ligand, and receptor. In general, the method can account for the complex hydrodynamic, translational-orientational diffusion aspects of ligand-docking dynamics as well as predict the actual or absolute rates of ligand binding. To test some of the underlying features of the method, simulations were conducted here for an artificially constructed spherical protein "made" from the real protein insulin. Excellent comparisons of simulation calculations of the so-called grand particle friction tensor to analytical values were obtained for this system when protein charge effects were neglected. When protein charges were included, we found anomalous results caused by the alteration of the spatial, microscopic structure of water proximal to the protein surface. Protein charge effects were found to be highly significant and consistent with the recent hypothesis of Hoppert and Mayer (Am Sci 1999;87:518-525) for charged macromolecules in water, which involves the formation of a "water dense region" proximal to the charged protein surface followed by a "dilute water region." We further studied the algorithm on a D-peptide/HIV capside protein system and demonstrated the algorithms utility to study the nonequilibrium docking dynamics in this contemporary problem. In general, protein charge effects, which alter water structural properties in an anomalous fashion proximal to the protein surface, were found to be much more important than the so-called hydrodynamic interaction effects between ligand and receptor. The diminished role of hydrodynamic interactions in protein systems allows for a much simpler overall dynamic algorithm for the nonequilibrium protein-docking process. Further studies are now underway to critically examine this simpler overall algorithm in analyzing the nonequilibrium protein-docking problem.     

Fluctuations, responses and energetics of molecular motors

A novel equality relating the rate of energy dissipation to a degree of violation of the fluctuation-response relation (FRR) in non-equilibrium Langevin systems is described. The FRR is a relation between the correlation function of the fluctuations and the response function of macroscopic variables. Although it has been established that the FRR holds in equilibrium, physical significance of violation of the FRR in non-equilibrium systems has been under debate. Recently, the authors have found that an extent of the FRR violation is related in a simple equality to the rate of energy dissipation into the environment in non-equilibrium Langevin systems. In this paper, we fully explain the FRR, the FRR violation, and the new equality with regard to a Langevin model termed a Brownian motor model, which is considered as a simple model of a biological molecular motor. Furthermore, applications of our result to experimental studies of molecular motors are discussed, and, as an illustration, we predict the value of a new time constant regarding the motion of a KIF1A, which is a kind of single-headed kinesin.     

Studies on the kinetics and mechanism of anation reactions of some six-coordinate complexes of chromium(III) in aqueous solution

Rates of stepwise anation of cis-Cr(ox)₂(H₂O₂)⁻ with SCN⁻/N₃⁻, Cr(acac)₂(H₂O)₂⁺ with SCN⁻ and Cr(atda)(H₂O)₂ with SCN⁻ have been investigated in weakly acidic aqueous solutions. Rate constants, k_I and k_II for the two steps in each system, are composite as k_x = k_x⁰+k_x^X[X⁻] (x = I, II; X⁻ = SCN⁻, N₃⁻). These rate constants have been evaluated also as the corresponding ΔH^≠ and ΔS^≠ values. The results obtained and the plausible I_d mechanism seem to suggest Cr---OOC bond dissociation (hence a strongly negative ΔS^≠) generating the transition state in each system with outer-sphere association forming the precursor complex in the X⁻ dependent paths.     

Dimensions,energetics, and denaturant effects of the protein unstructured state

Determining the energetics of the unfolded state of a protein is essential for understanding the folding mechanics of ordered proteins and the structure–function relation of intrinsically disordered proteins. Here, we adopt a coil‐globule transition theory to develop a general scheme to extract interaction and free energy information from single‐molecule fluorescence resonance energy transfer spectroscopy. By combining protein stability data, we have determined the free energy difference between the native state and the maximally collapsed denatured state in a number of systems, providing insight on the specific/nonspecific interactions in protein folding. Both the transfer and binding models of the denaturant effects are demonstrated to account for the revealed linear dependence of inter‐residue interactions on the denaturant concentration, and are thus compatible under the coil‐globule transition theory to further determine the dimension and free energy of the conformational ensemble of the unfolded state. The scaling behaviors and the effective θ‐state are also discussed.     

Electron exchange reaction of bis(terpyridine)cobalt re-examined by NMR

The rate of the electron transfer self-exchange reaction between bis(terpyridine) cobalt(III) and bis(terpyridine)cobalt(II) has been reexamined by proton NMR. The rate constant of 4×10² M⁻¹ s⁻¹ at 50 °C is dependent on the identity of the anion. Average activation parameters of 32 kJ mol⁻¹ and −96 J K⁻¹ mol⁻¹ are in agreement with previous measurements by other techniques. There is no evidence for either spin restrictions or non-adiabtaticity in this and related cobalt(III)/(II) electron exchange reactions. An alternative explanation is offered for the anomalously negative volumes of activation reported elsewhere.     

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction ³Zncyt + az(II) → Zncyt⁺ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrosatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.     

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Content and redox properties of electron-transfer components

Stable and well coupled Photosystem (PS) I-enriched vesicles, mainly derived from the chloroplast stroma lamellae, have been obtained by mild digitonin treatment of spinach chloroplasts. Optimal conditions for chloroplast solubilization are established at a digitonin/chlorophyll ratio of 1 ($\text{w}\text{w}$) and a chlorophyll concentration of 0.2 mM, resulting in little loss of native components. In particular, plastocyanin is easily released at higher digitonin/chlorophyll ratios. On the basis of chlorophyll content, the vesicles show a 2-fold enrichment in ATPase, chlorophyll-protein Complex I, P-700, plastocyanin and ribulose-1,5-bisphosphate carboxylase as compared to chloroplasts, in line with the increased activities of cyclic photophosphorylation and PS I-associated electron transfer as shown previously (Peters, A.L.J., Dokter, P., Kooij, T. and Kraayenhof, R. (1981) in Photosynthesis I (Akoyunoglou, G., ed.), pp. 691–700, Balaban International Science Services, Philadelphia). The vesicles have a low content of the light-harvesting chlorophyll-protein complex and show no PS II-associated electron transfer. Characterization of cytochromes in PS I-enriched vesicles and chloroplasts at 25°C and 77 K is performed using an analytical method combining potentiometric analysis and spectrum deconvolution. In PS I-enriched vesicles three cytochromes are distinguished: c-554 (E′₀ = 335 mV), b-559_LP (E′₀ = 32 mV) and b-563 (E′₀ = ? 123 mV); no b-559_HP is present (LP, low-potential; HP, high-potential). Comparative data from PS I vesicles and chloroplasts are consistent with an even distribution of the cytochrome b-563- cytochrome c-554 redox complex in the lateral plane of exposed and appressed thylakoid membranes, an exclusive location of plastocyanin in the exposed membranes and a dominant location of plastoquinone in the appressed membranes. The results are discussed in view of the lateral heterogeneity of redox components in chloroplast membranes.     

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

Horse heart cytochrome c and French bean plastocyanin are cross-linked one-to-one by a carbodiimide [Geren, L. M., Stonehuerner, J., Davis, D. J., & Millett, F. (1983) Biochim. Biophys. Acta 724, 62] in the same general orientation in which they associate electrostatically [King, G. C., Binstead, R. A., & Wright, P. E. (1985) Biochim. Biophys. Acta 806, 262]. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV vs NHE; the EPR spectra of the two metals are not perturbed by cross-linking. Four isomers of the covalent diprotein complex, which probably differ slightly from one another in the manner of cross-linking, are separated efficiently by cation-exchange chromatography. Stopped-flow spectrophotometric experiments with the covalent diprotein complex show that the presence of plastocyanin somewhat inhibits oxidation of ferrocytochrome c by [Fe(CN)6]3- and somewhat promotes oxidation of this protein by [Fe(C5H5)2]+. These changes in reactivity are explained in terms of electrostatic and steric effects. Pulse-radiolysis experiments with the electrostatic diprotein complex yield association constants of greater than or equal to 5 X 10(6) and 1 X 10(5) M-1 at ionic strengths of 1 and 40 mM, respectively, and the rate constant of 1.05 X 10(3) s-1, regardless of the ionic strength, for the intracomplex electron-transfer reaction. Analogous pulse-radiolysis experiments with each of the four isomers of the covalent diprotein complex, at ionic strengths of both 2 and 200 mM, show an absence of the intracomplex electron-transfer reaction. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.     

Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations

Many important proteins contain multiple domains connected by flexible linkers. Inter-domain motion is suggested to play a key role in many processes involving molecular recognition. Heteronuclear NMR relaxation is sensitive to motions in the relevant time scales and could provide valuable information on the dynamics of multi-domain proteins. However, the standard analysis based on the separation of global tumbling and fast local motions is no longer valid for multi-domain proteins undergoing internal motions involving complete domains and that take place on the same time scale than the overall motion.The complexity of the motions experienced even for the simplest two-domain proteins are difficult to capture with simple extensions of the classical Lipari-Szabo approach. Hydrodynamic effects are expected to dominate the motion of the individual globular domains, as well as that of the complete protein. Using Pin1 as a test case, we have simulated its motion at the microsecond time scale, at a reasonable computational expense, using Brownian Dynamic simulations on simplified models. The resulting trajectories provide insight on the interplay between global and inter-domain motion and can be analyzed using the recently published method of isotropic Reorientational Mode Dynamics which offer a way of calculating their contribution to heteronuclear relaxation rates. The analysis of trajectories computed with Pin1 models of different flexibility provides a general framework to understand the dynamics of multi-domain proteins and explains some of the observed features in the relaxation rate profile of free Pin1.     

Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae

Respiration in Saccharomyces cerevisiae is regulated by small proteins such as the respiratory supercomplex factors (Rcf). One of these factors (Rcf1) has been shown to interact with complexes III (cyt. bc₁) and IV (cytochrome c oxidase, CytcO) of the respiratory chain and to modulate the activity of the latter. Here, we investigated the effect of deleting Rcf1 on the functionality of CytcO, purified using a protein C-tag on core subunit 1 (Cox1). Specifically, we measured the kinetics of ligand binding to the CytcO catalytic site, the O₂-reduction activity and changes in light absorption spectra. We found that upon removal of Rcf1 a fraction of the CytcO is incorrectly assembled with structural changes at the catalytic site. The data indicate that Rcf1 modulates the assembly and activity of CytcO by shifting the equilibrium of structural sub-states toward the fully active, intact form.     

The kinetics and mechanisms of the complex formation and antioxidant behaviour of the polyphenols EGCg and ECG with iron(III)

(-)-Epigallocatechin-gallate ((-)-EGCg) and (-)-epicatechin-gallate ((-)-ECG) are important antioxidants which are found in green tea. The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with EGCg and ECG have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are consistent with a mechanism in which the 2:1 metal:ligand complex initially formed on reaction of iron(III) with the ligand subsequently decomposes in an electron transfer step. Complex formation takes place at two separate binding sites via coupled reactions. Rate constants of 4.28(+/-0.06) x 10(6) M(-2) s(-1) and 2.83(+/-0.04) x 10(6) M(-2) s(-1) have been evaluated for the reaction of monohydroxy Fe(OH)2+ species with EGCg and ECG, respectively while rate constants for of 2.94(+/-0.4) x 10(4) M(-2) s(-1) and 2.41(+/-0.25) x 10(4) M(-2) s(-1) have been evaluated for the reaction of Fe3+ species with EGCg and ECG, respectively. The iron(III) assisted decomposition of the initial iron(III) complex formed was also investigated and the rate constants evaluated. Both the complex formation and subsequent electron transfer reactions of iron(III) with EGCg and ECG were monitored using UV-visible spectrophotometry. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time dependant spectra. The results obtained show that one molecule of either EGCg or EGC is capable of reducing up to four iron(III) species, a fact which is consistent with the powerful antioxidant properties of the ligands.     

cDNA cloning,functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5′-terminal and 3′-terminal were obtained by RACE technique. The full-length cDNAthat encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.