Polymethionyl,-valyl and-glycyl derivatives of equine heart cytochromec heme octapeptide

Polymethionyl ((Met)_5·3-H8PT), polyvalyl ((Val)_4·4-H8PT) and polyglycyl ((Gly)_3·2-H8PT) derivatives of the heme octapeptide of equine heart cytochrome c were prepared with the aid of the N-carboxyl anhydrides of their respective amino acids. Only for the (Met)_5·3-H8PT did the γ-peak in the absorption spectrum of the reduced form shift from 412.5 nm, the value for the unsubstituted octapeptide, to 414 nm, the value close to the γ-peak of intact ferrocytochrome c. The γ-peak of the oxidized form of the octapeptide did not shift significantly from 397 nm. Amino acids attached to the N-terminal cysteine of the octapeptide, with methionine excepted, have little or no effect on the spectrum, and apparently cannot serve as either an intramolecular or intermolecular ligand for the iron. This result is in marked contrast to those previously obtained with the substituted cytochrome c heme undecapeptide (ref. 8) where all three derivatives had altered spectra. For the octapeptide, the ratio, absorbance of the γ-peak of the oxidized form to absorbance of the γ-peak of the reduced form, did not change appreciably for any of the derivatives, although when the reaction products from excess methionine anhydride were present (before centrifugation and dialysis) the ratio was lower and approached the ratio for cytochrome c.     

Cationic heme undecapeptide as stain for detecting glycosaminoglycans on cellulose acetate strips

A rapid and sensitive method has been developed for detecting glycosaminoglycans on cellulose acetate strips. The method is based on the electrostatic interactions between glycosaminoglycans and cationic heme undecapeptide. The complexes are identified by peroxidatic reaction using 3,3'-diaminobenzidine as H2 donor. The detection limit is 2.5-10 ng for sulfated glycosaminoglycans and 60 ng for hyaluronic acid. The sensitivity of this method is higher than of those using Alcian blue or toluidine blue (500 ng).     

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?     

A study of the electron transfer properties of the heme undecapeptide from cytochrome c by 1H nmr spectroscopy

Nuclear magnetic resonance (nmr) spectroscopy has been used to investigate the heme undecapeptide from cytochrome c. Assignments of resonances to specific residues have been made based on spin decoupling, redox titration, and the pH and temperature dependence of resonance lines. An outline structure is presented based on the assignments, secondary shift data, and the x-ray crystal structure of cytochrome c. An equation is derived to relate the width of an nmr line during a redox titration to the percentage of each oxidation state. Using this equation the self-exchange rate constant for electron transfer for the heme peptide is 1.3 x 10(7) M-1 sec-1 at 330 degrees K. Discussion of the self-exchange rate constants of cytochrome c, cytochrome c3, and cytochrome c551 is related to this constant for the heme undecapeptide.     

Interactions of cytochromec with phospholipid membranes

Summary Cytochromec added during the formation of lecithin-cardiolipin liquid crystals in 0.015m KCl is readily bound. After successive washings with 0.15m KCl, only about 50% of this bound cytochromec is removed. The remaining cytochromec is resistant to further salt extraction, and the amount of this cytochromec that is bound varies with the concentration of added cytochromec to a maximum binding ratio of 170, mole ratio cytochromec to phospholipid. This binding appears to be electrostatic; it is competitively inhibited by increasing the initial molarity of KCl from 0.015 to 0.10m. Binding of cytochromec is insignificant in the absence of cardiolipin, and is affected by varying the pH. Electron microscope studies of osmium tetroxide-stained thin sections show that the liquid crystals consist of vesicles, each of which contains a large number of concentric, alternating light and dense lines. The dense lines have been identified by other workers with the polar head groups of the phospholipids on the surface of a bilayer, and the light area represents the hydrophobic interior. The addition of cytochromec causes an average decrease in the number of lines per vesicle. It increases the center-to-center distance between two neighboring light or dense lines and the width of the dense lines. On the basis of this evidence and electrostatic binding, it is concluded that cytochromec is binding on the polar surfaces of the phospholipid bilayers comprising the liquid crystalline vesicles.     

Evolutionary aspects of cytochromec oxidase

The presence of additional subunits in cytochrome oxidase distinguish the multicellular eukaryotic enzyme from that of a simple unicellular bacterial enzyme. The number of these additional subunits increases with increasing evolutionary stage of the organism. Subunits I–III of the eukaryotic enzyme are related to the three bacterial subunits, and they are encoded on mito-chondrial DNA. The additional subunits are nuclear encoded. Experimental evidences are presented here to indicate that the lower enzymatic activity of the mammalian enzyme is due to the presence of nuclear-coded subunits. Dissociation of some of the nuclear-coded subunits (e.g., VIa) by laurylmaltoside and anions increased the activity of the rat liver enzyme to a value similar to that of the bacterial enzyme. Further, it is shown that the intraliposomal nucleotides influence the kinetics of ferrocytochromec oxidation by the reconstituted enzyme from bovine heart but not fromP. denitrificans. The regulatory function attributed to the nuclear-coded subunits of mammalian cytochromec oxidase is also demonstrated by the tissue-specific response of the reconstituted enzyme from bovine heart but not from bovine liver to intraliposomal ADP. These enzymes from bovine heart and liver differ in the amino acid sequences of subunits VIa, VIIa, and VIII. The results presented here are taken to indicate a regulation of cytochromec oxidase activity by nuclear-coded subunits which act like receptors for allosteric effectors and influence the catalytic activity of the core enzyme via conformational changes.     

Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidase (EPO), and lactoperoxidase (LPO), homologous members of the mammalian peroxidase superfamily, can all serve as catalysts for generating nitric oxide- (nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prosthetic groups that are ligated through a histidine nitrogen and use H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic and/or conformational changes in their heme pockets using a combination of rapid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe(III) state] causes immediate buildup of the corresponding Fe(II) complexes. Spectral changes indicate that two LPO-Fe(II) species are present in solution at equilibrium. Analyses of stopped-flow traces collected when EPO, MPO, or LPO solutions rapidly mixed with NO were accurately fit by single-exponential functions. Plots of the apparent rate constants as a function of NO concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed significantly lower affinity toward NO compared to Fe(III) forms, suggesting that heme reduction causes a dramatic change in the heme pocket electronic environment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-spin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward binding of diatomic ligands. Our results suggest that, upon reduction, the heme pocket of MPO collapses, LPO adopts two spectroscopically and kinetically distinguishable forms (one partially open and the other relatively closed), and EPO remains open.     

Conformation of horse heart ferricytochrome c. III. Comparative optical rotatory dispersion study of the protein with its derivative heme undecapeptide

The optical rotatory dispersion of horse heart ferricytochrome c and of a ferri heme undecapeptide have been determined under various conditions. Analysis of the Soret region makes it possible to characterize three different states of ferricytochrome c. the native state (superposition of a negative and a positive Cotton effect); an intermediate state (single positive Cotton effect whose magnitude Δ[M] is equal to 55,000); a denatured state (single positive Cotton effect whose magnitude Δ[M] is equal to 115,000) in which compared to both the native and intermediate states a more or less important decrease in helix content is observed. The optical rotatory dispersion spectra of the Soret region of the monomeric ferri heme undecapeptide is similar to that of denatured ferricytochrome c. The multiplicity of Cotton effects observed under certain conditions for the hemopeptide is a consequence, resulting from a polymerization, of intermolecular interactions. The comparison of the optical rotatory dispersion spectra of ferricytochrome c and the ferri heme undecapeptide indicates that in the intermediate state interactions remain between the heme group and the portion of the poly pep tide chain absent in the hemopeptide. These interactions disappear in the denatured state.     

Diffusion-controlled reaction kinetics of the binding of carbon monoxide to the heme undecapeptide of cytochrome c (microperoxidase 11) in high viscosity solvents

Glycosphingolipids from human plasma with Le^a, Le^b, and H-type 1 (Le^dH) Lewis-blood-group activity have been analyzed after permethylation by electron impact mass spectrometry using an indirectly heated direct insertion probe. The spectra obtained are compared with that of permethylated neo-lactotetraosyl ceramide (Gl-3) from human plasma. The fragmentation patterns presented show clearly, that Le^a and H-type 1 glycosphingolipids are ceramide pentasaccharides while Le^b is a ceramide hexasaccharide. All Lewis-blood-group-active compounds investigated produced ions specific for type 1 carbohydrate chains. It is therefore concluded, that all compounds are derivatives of lacto-N-tetraose. The obtained spectra support the following sequences: Hexose-1→3-hexosamine[4←1-deoxyhexose]-hexose-hexose ceramide for the Le^a derivatives; deoxyhexose-hexose-1→3-hexosamine4←1-deoxyhexose]-hexose-hexose ceramide for the Le^b derivatives; and deoxyhexose-hexose-1→3-hexosamine-hexose-hexose ceramide for all H-type 1 (Le^dH) derivatives. In the case of the H-type 1 glycosphingolipids four subfractions were analyzed separately. While all four fractions contained the same carbohydrate sequence, significant differences were observed in the ceramide residues. Specific fragmentation patterns indicate the presence of sphingosine, icosasphingosine, and 4-hydroxysphinganine besides normal, unsaturated, and hydroxylated fatty acids in all Lewis-blood-group-active glycolipids.     

A systematist looks at cytochromec

Summary The available data (as of June 1972) on amino-acid sequences of cytochromec are reviewed from the point of view of a traditional phylogenetic systematist. The Darwinian assumption, that phylogenetic changes in the sequence have been controlled by natural selection, is made, and some tentative phylogenetic and systematic conclusions are drawn. Attention is drawn to apparent correlations between substitutions at different points in the molecule. Suggestions for further investigations are made.     

Evolutionary change in invertebrate cytochromec

Summary Recently published amino acid sequences are compared to those of other cytochromesc. Molecular phylogenies constructed by using an ancestral sequence method are compared to the classical biological view of invertebrate evolution. Problems associated with the analysis of sequences of different chain lengths and of high variability are discussed, and the logistics of increasing the representation of key invertebrate phyla is assessed.     

Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1

We report the crystal structure of heme oxygenase from the pathogenic bacterium Neisseria meningitidis at 1.5 A and compare and contrast it with known structures of heme oxygenase-1 from mammalian sources. Both the bacterial and mammalian enzymes share the same overall fold, with a histidine contributing a ligand to the proximal side of the heme iron and a kinked alpha-helix defining the distal pocket. The distal helix differs noticeably in both sequence and conformation, and the distal pocket of the Neisseria enzyme is substantially smaller than in the mammalian enzyme. Key glycine residues provide the flexibility for the helical kink, allow close contact of the helix backbone with the heme, and may interact directly with heme ligands.     

Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.     

Functional binding of cardiolipin to cytochromec oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa ₃ complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K _d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [¹⁴C]-acetyl-cardiolipin (K _d <0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.     

The cytochromec oxidase from the yeastCandida parapsilosis

In the yeastCandida parapsilosis, the proteins encoded by mitochondrial DNA are different in number and size from those ofSaccharomyces cerevisiae. Nevertheless, the purified cytochromec oxidase fromCandida parapsilosis shows kinetic properties similar to those ofSaccharomyces cerevisiae.     

Succinylated bovine heart mitochondrial cytochromec oxidase

Cytochromec oxidase was prepared by sequential extraction of bovine heart muscle submitochondrial particles with sodium deoxycholate, followed by fractional precipitation with ammonium sulfate and chromatography on Sephadex G-75. The resulting preparation had typical absorption spectra, an activity of 1.28 sec^–1 (mg protein)^–1 (3 ml)^–1 in deoxycholate or 4.13 sec^–1 (mg protein)^–1 (3 ml)^–1 in 0.5% Tween 80, and a minimum molecular weight of 120,000 daltons as calculated from the heme content and the total protein. Amino acid analyses of nine preparations yielded a molecular weight per heme of 86,500 daltons. The net charge was calculated to be +8.7 at pH 7.0. Succinylation of cytochromec oxidase in the presence of 500 molar excess of succinic anhydride produced a soluble preparation having a negative charge at neutral pH. The modified enzyme was highly autoxidizable and had little or no activity toward ferrocytochromec as a substrate. Its averageS _20,w was 5.8 and its apparentD was 4.0 × 10^–7 cm² sec^–1, from which a molecular weight of 126,000 daltons was calculated. This size of enzyme is considered to be that of the monomer, because the value is practically the same as the minimum molecular weight reported herein, and since it is approximately onehalf the value obtained in our laboratory (and in others) for the unmodified enzyme.     

Deaggregation and proteolytic modification of a galactosyltransferase of Poterioochromonas malhamensis

We isolated hybridomas that produced monoclonal antibodies specific for the UDP-galactose: sn -glycerol-3-phosphate α-D-galactosyltransferase (IFP synthase, EC 2.4.1.96), an enzyme involved in the volume regulation of Poterioochromonas malhamensis Peterfi. Western blotting of native gradient gels with the most reactive antibody S 162 revealed several immunoreactive proteins in crude homogenates suggesting the occurrence of multiple molecular mass species of the galactosyltransferase. The amount of the presumed enzyme monomer (64 kDa under native conditions) was strongly increased by a pH shift of crude homogenates from pH 8 to 6. During activation of the galactosyltransferase in the cell homogenate and also by shrinking the cells, the presumed enzyme monomer appeared to be proteolytically degraded generating stepwise products of 52 and 40 kDa. We assume that the proteolytically processed enzyme becomes highly active, but is very susceptible to further proteolytic degradation.