Site Of Cytochrome Research Articles

Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl

Cytochrome c oxidase isolated from bovine heart was crystallized in the fully reduced carbon monoxide (CO)-bound form. To evaluate the structure of the O2 reaction site in crystals and in solution, the bound C-O stretch infrared band in protein crystals was compared with the band for protein solution. In solution, the C-O stretch band could be deconvoluted into two extremely narrow bands, one at 1963.6 cm-1 with delta v1/2 = 3.4 cm-1 of 60% Gaussian/40% Lorentzian character represented 86% of the total band area and the other at 1960.3 cm-1 with delta v1/2 = 3.0 cm-1 of 47% Gaussian/53% Lorentzian character represented 14% of the total band area. The crystals exhibited two deconvoluted C-O infrared bands having very similar band parameters with those in solution. These findings support the presence of two structurally similar conformers in both crystals and solution. Thus crystallization of this enzyme does not affect the structure at the CO-binding site to as great extent as has been noted for myoglobin and hemoglobin carbonyls, indicating that the active (CO- or O2-binding) site of cytochrome c oxidase must be conformationally very stable and highly ordered compared to other hemoproteins such as hemoglobin.

EPR of nitrous oxide reductase: A model for the EPR detectable site in cytochrome C oxidase

EPR of nitrous oxide reductase: A model for the EPR detectable site in cytochrome C oxidase

Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase.

Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330). As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP. Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide. The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of [14C]phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex. The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products. Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme. Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity. Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity. These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.

Multifrequency EPR evidence for a bimetallic center at the Cu A site in cytochrome c oxidase

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes. C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g-values are calculated for g z and g x at four frequencies. No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.

RNA sequence and the nature of the Cu A-binding site in cytochrome c oxidase

For cytochrome c oxidase subunit II (COXII), DNA and protein sequences suggest that Met-207 (bovine numbering) is conserved in all species except plants. Sequencing of plant mitochondrial COXII mRNAs now indicates that Met-207 is also conserved among plants as a result of a C-to-U type of RNA editing. Considering the strict evolutionary conservation of Met-207 and the homology of COXII to type I (blue) copper proteins and nitrous oxide reductase, we propose a model in which Met-207 is associated with the Cu A-binding site (along with Cys-196, Cys-200 and His-204) and plays a role in determining its reduction potential and stability.

The kinetics of electron entry in cytochromec oxidase

The kinetics of electron entry in beef heart cytochrome c oxidase have been studied by stopped-flow spectroscopy following chemical modification of the CuA site with mercurials. In this derivative CuA is no longer reducible by cytochrome c while cytochrome alpha may accept electrons from the latter with rates comparable to the native enzyme. The results indicate that CuA is not the exclusive electron entry site in cytochrome c oxidase.

ChemInform Abstract: Heterocyclic o‐Chloroaldehydes as Synthons in Organic and Inorganic Sulfur Chemistry

Abstract Many heterocyclic o-chloroaldehydes are easy to prepare, for example via the Vilsmeier chloroformylation reaction. Due to the electron withdrawing effect of the formyl group, the halogen atom is normally a nucleofuge. This can be used to prepare a range of o-substituted heterocyclic aldehydes showing interesting rearrangement reactions. Such types of compounds are also useful as starting materials for the preparation of annelated heterocyclic systems. We have demonstrated that heterocyclic mercapto groups can be protected as t-butylthioethers. Furthermore, we have found that thiolation of heterocyclic systems containing reactive halogen substituents can conveniently be carried out via the easily prepared t-butylsulfides. This method has been used in the preparation of a number of N2S2-ligands. The resulting transition metal N2S2-complexes have been used in the study of model systems for one of the active sites in cytochrome c oxidase. The method also gives access to polymeric heterocyclic multisu...

Identification by ENDOR of Trp 191 as the Free-Radical Site in Cytochrome c Peroxidase Compound ES

The chemical identity of the amino acid free-radical site that represents one of the two oxidizing equivalents stored in the H2O2-oxidized intermediate (compound ES) of the mitochondrial heme enzyme, cytochrome c peroxidase (CcP) has been sought for almost a quarter of a century. Site-directed mutagenesis alone cannot yield this answer. Low-temperature 35-gigahertz (Q-band) electron nuclear double resonance (ENDOR) spectroscopy was used to examine compound ES prepared from proteins containing specifically deuterated methionine or tryptophan, as well as the amino acid replacement Trp51----Phe. The results definitely identify the site of the radical in compound ES as tryptophan, most likely Trp191.

ChemInform Abstract: Molecular Recognition in Cytochrome P‐450: Alteration of Regioselective Alkane Hydroxylation via Protein Engineering.

AbstractThe variation of three active sites in cytochrome P‐450 affords changes in the regioselectivity of hydroxylation of camphor (II) and its demethylated derivatives such as (IV).

Pseudomonas stutzeri N2O reductase contains CuA-type sites.

N2O reductase (N2O----N2) is the terminal enzyme in the energy-conserving denitrification pathway of soil and marine denitrifying bacteria. The protein is composed of two identical subunits and contains eight copper ions per enzyme molecule. The magnetic circular dichroism spectrum of resting (oxidized) N2O reductase is strikingly similar to the magnetic circular dichroism spectrum of the CuA site in mammalian cytochrome c oxidase [Greenwood, C., Hull, B. C., Barber, D., Eglinton, D. G. & Thomson, A. J. (1983) Biochem. J. 215, 303-316] and is unlike the magnetic circular dichroism spectra of all other biological copper chromophores obtained to date. Sulfur (or chlorine) scatterers are required to fit the copper extended x-ray absorption fine structure data of both the oxidized and reduced forms of N2O reductase. Satisfactory fits require a Cu-N or Cu-O [denoted Cu-(N, O)] interaction at 2.0 A, a Cu-(S, Cl) interaction at 2.3 A and an additional Cu(S, Cl) interaction at approximately 2.6 A (oxidized) or approximately 2.7 A (reduced). Approximately eight sulfur ions (per eight copper ions) at approximately 2.3 A are required to fit the extended x-ray absorption fine structure data for both the oxidized and reduced N2O reductase. The 2.3-A Cu-(S, Cl) distance is nearly identical to that previously determined for the CuA site in cytochrome c oxidase. A 2.6-2.7 A Cu-(S, Cl) interaction is also present in resting and fully reduced cytochrome c oxidase. Comparison of the N2O reductase sequence, determined by translating the structural NosZ gene, with cytochrome c oxidase subunit II sequences from several sources indicates that a Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Ser-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-His stretch is highly conserved. This sequence contains three of the probable ligands (two cysteines and one histidine) in a CuA-type site. Collectively these data establish that Pseudomonas stutzeri N2O reductase contains CuA-type sites.

Heterocyclico-Chloroaldehydes as Synthons in Organic and Inorganic Sulfur Chemistry

Abstract Many heterocyclic o-chloroaldehydes are easy to prepare, for example via the Vilsmeier chloroformylation reaction. Due to the electron withdrawing effect of the formyl group, the halogen atom is normally a nucleofuge. This can be used to prepare a range of o-substituted heterocyclic aldehydes showing interesting rearrangement reactions. Such types of compounds are also useful as starting materials for the preparation of annelated heterocyclic systems. We have demonstrated that heterocyclic mercapto groups can be protected as t-butylthioethers. Furthermore, we have found that thiolation of heterocyclic systems containing reactive halogen substituents can conveniently be carried out via the easily prepared t-butylsulfides. This method has been used in the preparation of a number of N2S2-ligands. The resulting transition metal N2S2-complexes have been used in the study of model systems for one of the active sites in cytochrome c oxidase. The method also gives access to polymeric heterocyclic multisu...

Some spectroscopic views of the CuA site in cytochrome c oxidase preparations.

Some spectroscopic views of the CuA site in cytochrome c oxidase preparations.

The Cytochrome‐c Binding Surface of Reaction Centers from Rhodobacter sphaeroides

AbstractIn this paper, we describe the water‐exposed, periplasmic surface of the bacterial photosynthetic reaction center from Rhodobacter sphaeroides R‐26. This surface contains binding sites for c‐type cytochromes. The approximate two‐fold structural symmetry is evident in the folding of the L and M subunits of the reaction center on this surface. In spite of this, the distribution of charged residues shows a marked deviation from symmetry. One group of charged residues is distributed symmetrically. For this group, equivalently charged residues are located at symmetry‐related positions on both L and M subunits. However, charged residues are also found at positions which do not follow the two‐fold symmetry. Seven charged residues appear on the L and M subunits at positions which do not have a charged residue at the corresponding position on the other subunit. These symmetry‐breaking charged residues appear in a single region, or “patch”, on the surface of the reaction center. We propose that this patch may function as a site for cytochrome c docking. A model of the Rb. sphaeroides cytochrome c2‐reaction center electron transfer complex has been built. Preliminary energy calculations show that this configuration is at least a local energy minimum.

Substrate-Specific Effect of Oleic Acid and Serum from Patients with Reye's Syndrome on Respiratory Control Ratio of Rat Liver Mitochondria

The effects of oleic acid and the serum from two patients with Reye's syndrome (RS-serum) on the respiratory control ratio (RCR) of rat liver mitochondria were studied; the reaction site of each reagent was determined by using various substrates for the respiratory chain. The results were as follows: 1. Pre-incubation of rat liver mitochondria with oleic acid induced a decrease in RCR when succinate or ascorbate+cytochrome c were employed as substrates. 2. Under the identical conditions, RS-serum induced a significant decrease in RCR with ascorbate+cytochrome c. 3. When KCN was added to the assay medium, both oxygen consumption rate and RCR were decreased as expected in oxidizing ascorbate+cytochrome c. But the effect of KCN was less obvious when pyruvate+L-malate were employed as substrates. The latter phenomenon might suggest that the rate-limiting step in the electron transport system from NADH to O2 resided on the substrate side and not on the side between coenzyme Q and O2.Among the substrates (succinate, pyruvate+L-malate, ascorbate+N, N, N′, N′-tetramethyl-phenylenediamine, ascorbate+cytochrome c) tested, the strongest effect was observed when ascorbate+cytochrome c were employed as substrates. These results suggest that both oleic acid and RS-serum showed similar substrate-specific inhibition of RCR, predominantly at the site of cytochrome c oxidase. Thus, a localized effect of oleic acid or RS-serum on the mitochondrial respiratory chain became apparent.

Multiple electron-transfer sites in cytochrome c oxidase?

Multiple electron-transfer sites in cytochrome <i>c</i> oxidase?

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome- c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s −1; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s −1 can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10 −4 to 10 −5-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.

Distribution of Diaminobenzidine Reaction Products in Zoospores of Phytophthora palmivora

Because microbodies in many filamentous fungi fail to stain with the 3,3′-diaminobenzidine (DAB) technique, the subcellular localization of DAB reaction products at two pHs was explored in an electron microscopic study of zoospores of Phytophthora palmivora. At pH 9.2, but not at pH 7.2, DAB reaction product was in the matrix of U-bodies and microbodies, indicating that both organelles contained catalasc. In contrast to a homogeneous distribution in microbodies, reaction product was limited to the core of U-bodies and absent from their halo and shell regions. At both pH 7.2 and 9.2, reaction product filled mitochondrial cristae, the site of cytochrome c oxidase. In addition to the organellar compartmentalization of reaction product, DAB reactions at pH 9.2 and 7.2 enhanced the prominence of an asymmetry in the trilamellar structure of the zoospore plasma membrane and the appearance of a cell coat covering the plasma membrane and flagellar sheath. This coat formed during zoosporogenesis, and DAB reactions intensified cell coat material that lined the inner surfaces of membranes involved in cytoplasmic cleavage. Since no reaction product formed in tissues incubated in reaction media to which sodium azide was added or DAB eliminated, reaction products were enzymatically produced. Because the DAB reaction highlights plasma membrane asymmetry and the presence of a cell coat, this technique should be useful in morphologically marking plasma membranes, enabling their recognition during zoosporogenesis or in isolated subcellular fractions of this fungus.

Distribution of Diaminobenzidine Reaction Products in Zoospores ofPhytophthora Palmivora

Because microbodies in many filamentous fungi fail to stain with the 3,3'-diaminobenzidine (DAB) technique, the subcellular localization of DAB reaction products at two pHs was explored in an electron microscopic study of zoospores of Phytophthora palmivora. At pH 9.2, but not at pH 7.2, DAB reaction product was in the matrix of U-bodies and microbodies, indicating that both organelles contained catalase. In contrast to a homogeneous distribution in microbodies, reaction product was limited to the core of U-bodies and absent from their halo and shell regions. At both pH 7.2 and 9.2, reaction product filled mitochondrial cristae, the site of cytochrome c oxidase. In addition to the organellar compartmentalization of reaction product, DAB reactions at pH 9.2 and 7.2 enhanced the prominence of an asymmetry in the trilamellar structure of the zoospore plasma membrane and the appearance of a cell coat covering the plasma membrane and flagellar sheath. This coat formed during zoosporogenesis, and DAB reactions intensified cell coat material that lined the inner surfaces of membranes involved in cytoplasmic cleavage. Since no reaction product formed in tissues incubated in reaction media to which sodium azide was added or DAB eliminated, reaction products were enzymatically produced. Because the DAB reaction highlights plasma membrane asymmetry and the presence of a cell coat, this technique should be useful in morphologically marking plasma membranes, enabling their recognition during zoosporogenesis or in isolated subcellular fractions of this fungus.

Structural aspects of the copper sites in cytochrome c oxidase. An X-ray absorption spectroscopic investigation of the resting-state enzyme.

Copper K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the structural details of the coordination environment of the copper sites in eight resting-state samples of beef heart cytochrome c oxidase prepared by different methods. The unusual position and structure of the resting-state copper edge spectrum can be adequately explained by the presence of sulfur-containing ligands, with a significant amount of S----Cu(II) charge transfer (i.e., a covalent site). Quantitative curve-fitting analysis of the copper extended X-ray absorption fine structure (EXAFS) data indicates similar average first coordination spheres for all resting-state samples, regardless of preparation method. The average coordination sphere (per 2 coppers) mainly consists of 6 +/- 1 nitrogens or oxygens at an average Cu-(N,O) distance of 1.99 +/- 0.03 A and 2 +/- 1 sulfurs at an average Cu-S distance of 2.28 +/- 0.02 A. Quantitative curve-fitting analysis of the outer shell of the copper EXAFS indicates the presence of a Cu...Fe interaction at a distance of 3.00 +/- 0.03 A. Proposed structures of the two copper sites based on these and other spectroscopic results are presented, and differences between our results and those of other published copper XAS studies [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] are discussed.

Topology and neighbor analysis of the photosynthetic reaction center from Rhodopseudomonas sphaeroides.

The subunit arrangement of the reaction center complex (RC) of Rhodopseudomonas sphaeroides was studied by chemical modification with four different cross-linking reagents using purified RC in lauryldimethylamine oxide, RC incorporated into liposomes, and intact chromatophore membranes, from which RCs are isolated. The RC of R. sphaeroides is composed of three polypeptide subunits, H, M, and L, apparent molecular mass as determined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 28,000, 24,000, and 21,000, respectively. The intra-complex products produced, were found to contain the polypeptides H-M-L, H-M, H-L, and M-L linked together. In addition, the cross-linking of cytochrome c to solubilized and membrane-bound RCs was observed with all four reagents. The products were found to be only a cytochrome c linked to either the M or L polypeptide. These results indicate that a portion of the L and M subunits of the RC must be exposed in situ on the periplasmic surface of the membrane near a binding site for cytochrome c on the RC, and all three subunits must be in close proximity to one another.