The pH dependence of the stereochemistry around the heme group in NO–cytochrome c (horse heart)

The electronic absorption, EPR and MCD spectra of NO derivatives of both ferrous and ferric cytochrome c (horse heart) have been measured in the pH region 2.0 to 12.9, in order to elucidate the pH dependence of the stereochemistry around the heme group. The reaction products of NO with ferrous cytochrome c in equilibrium were as follows: in the region 2.0 ⩽ pH ⩽ 5.3, NO–ferrous cytochrome c; in the region 5.3 < pH ⩽ 11.0, a mixture of NO–ferrous cytochrome c and native ferrous cytochrome c; at pH 12.0, NO–ferrous cytochrome c. At pH 2.0, the NO–ferrous cytochrome c contained a five-coordinate nitrosylheme as the major component and a six-coordinate species as the minor component, and at the order pH values it contained only the six-coordinate species. The reaction products of NO with ferric cytochrome c in equilibrium were as follows: in the region 2.0 ⩽ pH ⩽ 7.2, NO–ferric cytochrome c with six-coordinate nitrosylheme; in the region 7.2 < pH ⩽ 11.0, a mixture of NO–ferrous cytochrome c and native ferrous cytochrome c; at pH 12.0, NO–ferrous cytochrome c. Thus, the reaction of NO with ferric cytochrome c results in the formation of NO–ferrous cytochrome c, which is a typical case of reductive nitrosylation.     

Reductive activation of the heme iron–nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study

Cytochrome c nitrite reductase catalyzes the six-electron, seven-proton reduction of nitrite to ammonia without release of any detectable reaction intermediate. This implies a unique flexibility of the active site combined with a finely tuned proton and electron delivery system. In the present work, we employed density functional theory to study the recharging of the active site with protons and electrons through the series of reaction intermediates based on nitrogen monoxide [Fe(II)-NO(+), Fe(II)-NO·, Fe(II)-NO(-), and Fe(II)-HNO]. The activation barriers for the various proton and electron transfer steps were estimated in the framework of Marcus theory. Using the barriers obtained, we simulated the kinetics of the reduction process. We found that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers (PCETs) or in the form of three consecutive elementary steps involving reduction, PCET, and protonation. Kinetic simulations revealed the recharging through two PCETs to be a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO· intermediate. The radical transfer role for the active-site Tyr(218), as proposed in the literature, cannot be confirmed on the basis of our calculations. The role of the highly conserved calcium located in the direct proximity of the active site in proton delivery has also been studied. It was found to play an important role in the substrate conversion through the facilitation of the proton transfer steps.     

The complex of cytochrome c and cytochrome c peroxidase: the end of the road?

Cytochrome c (Cc) and cytochrome c peroxidase (CcP) form a physiological complex in the inter-membrane space of yeast mitochondria, where CcP reduces hydrogen peroxide to water using the electrons provided by ferrous Cc. The Cc-CcP system has been a popular choice of study of interprotein biological electron transfer (ET) and in understanding dynamics within a protein-protein complex. In this review we have charted seven decades of research beginning with the discovery of CcP and leading to the latest functional and structural work, which has clarified the mechanism of the intermolecular ET, addressed the putative functional role of a low-affinity binding site, and identified lowly-populated intermediates on the energy landscape of complex formation. Despite the remarkable attention bestowed on this complex, a number of outstanding issues remain to be settled on the way to a complete understanding of Cc-CcP interaction.     

Tyrosine triad at the interface between the Rieske iron–sulfur protein,cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus

A triad of tyrosine residues (Y152–154) in the cytochrome c₁ subunit (C1) of the Rhodobacter capsulatus cytochrome bc₁ complex (BC1) is ideally positioned to interact with cytochrome c₂ (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations—A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.     

Solution conformations of the pituitary opioid peptide dynorphin-(1–13)

Circular dichroism spectra have been measured for dynorphin-(1–13) in water and in solutions of sodium dodecyl sulfate and L-α-lysophosphatidylcholine (palmitoyl). Spectra in water have the features expected for a peptide containing little, if any, order. Small changes are brought about by L-α-lysophosphatidylcholine (palmitoyl), but the resulting spectrum retains the characteristics expected for a random coil. In contrast, sodium dodecyl sulfate produces significant changes which are those expected for induction of α helical content. Quantitative analysis of the circular dichroism spectra suggests the conformation changes from about 5% helix in water to 17% helix in sodium dodecyl sulfate. These results from experiment are in excellent agreement with those obtained from our formulation of the configuration partition function. This formulation predicts a change in helical content from 1% to 19%. The ordering influence is felt most strongly by those residues immediately following the enkephalin sequence.     

The binding interface of cytochrome c and cytochrome c₁ in the bc₁ complex: rationalizing the role of key residues

The interaction of cytochrome c with ubiquinol-cytochrome c oxidoreductase (bc₁ complex) has been studied for >30 years, yet many aspects remain unclear or controversial. We report the first molecular dynamic simulations of the cyt c-bc₁ complex interaction. Contrary to the results of crystallographic studies, our results show that there are multiple dynamic hydrogen bonds and salt bridges in the cyt c-c₁ interface. These include most of the basic cyt c residues previously implicated in chemical modification studies. We suggest that the static nature of x-ray structures can obscure the quantitative significance of electrostatic interactions between highly mobile residues. This provides a clear resolution of the discrepancy between the structural data and functional studies. It also suggests a general need to consider dynamic interactions of charged residues in protein-protein interfaces. In addition, a novel structural change in cyt c is reported, involving residues 21-25, which may be responsible for cyt c destabilization upon binding. We also propose a mechanism of interaction between cyt c₁ monomers responsible for limiting the binding of cyt c to only one molecule per bc₁ dimer by altering the affinity of the cytochrome c binding site on the second cyt c₁ monomer.     

How does heme axial ligand deletion affect the structure and the function of cytochrome b(562)?

We have recently generated a new mutant of cytochrome b(562) (cytb(562)) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb(562)). The M7A cytb(562) can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb(562), thermal transition analyses of M7A cytb(562) were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb(562) is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb(562) was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb(562), whereas the functional conversion of cytb(562) is successfully achieved by the loose folding together with the open coordination site.     

Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s).     

Identification of the minimum pharmacophore of lipid–phosphatidylserine (PS) binding peptide–peptoid hybrid PPS1D1

We previously reported a unique peptide–peptoid hybrid, PPS1 that specifically recognizes lipid–phosphatidylserine (PS) and a few other negatively charged phospholipids, but not neutral phospholipids, on the cell membrane. The dimeric version of PPS1, i.e., PPS1D1 triggers strong cancer cell cytotoxicity and has been validated in lung cancer models both in vitro and in vivo. Given that PS and other negatively charged phospholipids are abundant in almost all tumor microenvironments, PPS1D1 is an attractive drug lead that can be developed into a globally applicable anti-cancer agent. Therefore, it is extremely important to identify the minimum pharmacophore of PPS1D1. In this study, we have synthesized alanine/sarcosine derivatives as well as truncated derivatives of PPS1D1. We performed ELISA-like competitive binding assay to evaluate the PS-recognition potential and standard MTS cell viability assay on HCC4017 lung cancer cells to validate the cell cytotoxicity effects of these derivatives. Our studies indicate that positively charged residues at the second and third positions, as well as four hydrophobic residues at the fifth through eighth positions, are imperative for the binding and activity of PPS1D1. Methionine at the first position was not essential, whereas the positively charged Nlys at the fourth position was minimally needed, as two derivatives that were synthesized replacing this residue were almost as active as PPS1D1.     

Mechanistic insights into the superoxide–cytochrome c reaction by lysine surface scanning

This study summarizes results which have been obtained by a mutational study of human cytochrome c. The protein can be used as a recognition element in analytical assays and biosensors for superoxide radicals since ferricytochrome c reacts with superoxide to form ferrocytochrome c and oxygen. Here lysine mutagenesis of the distal surface (i.e., of exposed residues around the Met80 axial ligand) of human cytochrome c was pursued to evaluate the effect of the surface charges on the reaction rate with the superoxide anion radical and on the redox properties of the heme protein. The latter feature is particularly relevant when the protein is immobilized on a negatively charged self-assembled monolayer on an electrode to be used as a biosensor. The observed effects of the mutations are rationalized through structural investigations based on solution NMR spectroscopy and computational analysis of the surface electrostatics. The results suggest the presence of a specific path that guides superoxide toward an efficient reaction site. Localized positive charges at the rim of the entry channel are effective in increasing the reaction rate, whereas diffused positive charges or charges far from this area are not effective or are even detrimental, resulting in a misguided approach of the anion to the protein surface.     

Do photosynthetic bacteria contain cytochrome c1?

A method is described for characterizing, c-type cytochromes in bacterial membrane preparations according to molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Applied to the photosynthetic bacterium Rhodopseudomonas sphaeroides this technique is used, together with spectroscopic measurements, to demonstrate that a membrane-bound cytochrome c of mol.wt. 30000 is active in photosynthetic electron transport in addition to the well-known soluble cytochrome, cytochrome c2. The membrane cytochrome has a midpoint potential (E'0) at pH 7 of +290 mV, as compared with +360 mV for purified cytochrome c2. Its alpha-band has a peak near 552 nm, as compared with 550 nm for cytochrome c2. Evidence is presented that chromatophores contain roughly equal amounts of the two cytochromes.     

How rapid are the internal reactions of the ubiquinol:cytochrome c 2 oxidoreductase?

The temperature dependence of the partial reactions leading to turn-over of the UQH₂:cyt c ₂ oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c ₁ and c ₂, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Q_z-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole^–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole^–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)₂ P870, primary donor of the photochemical reaction center - b/c ₁ complex ubiquinol: cytochrome c ₂ oxidoreductase - cyt b _H cytochrome b-561 or higher potential cytochrome b - cyt b _L cytochrome b-566, or low potential cytochrome b - cyt c ₁, cyt c ₂, cyt c _t cytochromes c ₁ and c ₂, and total cytochrome c (cyt c ₁ and cyt c ₂) - Fe.S Rieske-type iron sulfur center, Q - QH₂ ubiquinone, ubiquinol - Q_z, Q_zH₂, Q_z ^– ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site - Q_z-site ubiquinol oxidizing site (also called Q_o-(outside) - Q_o (Oxidizing) - Q_P (Positive proton potential) site) - Q_c-site uubiquinone reductase site (also called the Q_i-(inside) - Q_R (Reducing), or - Q_N (Negative proton potential) site) - UHDBT 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol     

Effect of sol–gel encapsulation on the unfolding of ferric horse heart cytochrome c

Electronic absorption and resonance Raman spectra of ferric cytochrome c embedded in wet silica gels, in the presence of guanidine HCl as unfolding agent, between pH 0.35 and 7.0 are presented. The data clearly show that the ferric form of the protein encapsulated in sol–gel preserves its active site conformation. However, the spectra of the unfolded embedded protein are different from the corresponding spectra in solution suggesting that a strong interaction between the protein and the sol–gel takes place upon unfolding. The unfolding process mainly depends on the interaction between the exposed positive charges of the unfolded protein and the negatively charged functional groups of the silica surfaces. While this interaction partially stabilizes the protein in its native structure even at very acidic pH, in the presence of denaturants it has the opposite effect, causing mainly the weakening of both the heme-protein and the heme-ligand interactions.     

Solution structure of the human CD4 (403–419) receptor peptide

The cytoplasmic part of CD4 is known to be essential for the interaction with the human immunodeficiency virus type 1 proteins Vpu and Nef. The 17 amino acid synthetic peptide CD4 (403–419) with the amino acid sequence of the membrane proximal part of the cytoplasmic domain of the human CD4 receptor was structurally investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The average -helical content of the peptide could be estimated to be around 25%. Chemical shift index analysis and the connectivity pattern in nuclear Overhauser enhancement spectra located the -helical part of the peptide from Gln403 to Arg412. It may be speculated that this amphipathic -helix is the contact region with the Vpu and Nef proteins.The authors thank Prof. F.X. Schmid for help with the CD spectra.     

M?ssbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus

Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase. We have studied 87Fe-enriched samples with M?ssbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide. The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes. The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with M?ssbauer parameters quite similar to those of deoxymyoglobin. Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes. The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state. We have examined the oxidized enzyme from two different preparations. Both had good activity and identical optical and EPR spectra. The M?ssbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations. Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.     

Synthesis of oligonucleotide-peptide PEG-conjugated: the EGG (oligonucleotide)-chicken (peptide) dilemma?

The synthesis of a peptide-PEG-oligonucleotide chimera is compared when starting from the peptide or from the oligonucleotide sequence.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Structure of the mammalian antimicrobial peptide Bac7(1–16) bound within the exit tunnel of a bacterial ribosome

Proline-rich antimicrobial peptides (PrAMPs) produced as part of the innate immune response of animals, insects and plants represent a vast, untapped resource for the treatment of multidrug-resistant bacterial infections. PrAMPs such as oncocin or bactenecin-7 (Bac7) interact with the bacterial ribosome to inhibit translation, but their supposed specificity as inhibitors of bacterial rather than mammalian protein synthesis remains unclear, despite being key to developing drugs with low toxicity. Here, we present crystal structures of the Thermus thermophilus 70S ribosome in complex with the first 16 residues of mammalian Bac7, as well as the insect-derived PrAMPs metalnikowin I and pyrrhocoricin. The structures reveal that the mammalian Bac7 interacts with a similar region of the ribosome as insect-derived PrAMPs. Consistently, Bac7 and the oncocin derivative Onc112 compete effectively with antibiotics, such as erythromycin, which target the ribosomal exit tunnel. Moreover, we demonstrate that Bac7 allows initiation complex formation but prevents entry into the elongation phase of translation, and show that it inhibits translation on both mammalian and bacterial ribosomes, explaining why this peptide needs to be stored as an inactive pro-peptide. These findings highlight the need to consider the specificity of PrAMP derivatives for the bacterial ribosome in future drug development efforts.