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Inhibition of androgen biosynthesis is clinically effective for treating androgen-responsive prostate cancer. Abiraterone is a clinical first-in-class inhibitor of cytochrome P450 17A1 (CYP17A1) required for androgen biosynthesis. However, abiraterone also causes hypertension, hypokalemia, and edema, likely due in part to off-target inhibition of another steroidogenic cytochrome P450, CYP21A2. Abiraterone analogs were designed based on structural evidence that B-ring substituents may favorably interact with polar residues in binding CYP17A1 and sterically clash with residues in the CYP21A2 active site. The best analogs increased selectivity of CYP17A1 inhibition up to 84-fold compared with 6.6-fold for abiraterone. Cocrystallization with CYP17A1 validated the intended new contacts with CYP17A1 active site residues. Docking these analogs into CYP21A2 identified steric clashes that likely underlie decreased binding and CYP21A2 inhibition. Overall, these analogs may offer a clinical advantage in the form of reduced side effects. [ABSTRACT FROM AUTHOR]

Protein crystallization remains a serious bottleneck to structure determination by X-ray diffraction methods. Compounds acting as "molecular glue" provide a promising strategy to overcome this bottleneck. Such molecules interact via noncovalent bonds with two or more protein surfaces to promote lattice formation. Here, we report a 1.5 Å resolution crystal structure of lysine-rich cytochrome c complexed with p-phosphonatomethyl-calix[4]arene (pmclx4). Evidence for complex formation in solution was provided by NMR studies. Similar to p-sulfonato-calix[4]arene (sclx4), the cavity of pmclx4 entrapped a single lysine side chain. Interesting features of protein recognition by the phosphonate substituents were identified in the crystal structure. A new calixarene binding site was identified at Lys54. The electron density at this site indicated two distinct calixarene conformers, suggesting a degree of ligand mobility. The role of pmclx4 in protein crystal packing (molecular glue and patchy particle model) as well as differences in protein-binding with respect to sclx4 are discussed. [ABSTRACT FROM AUTHOR]

Hongkonoids A-D (1-4), the first example of ascorbylated terpenoids featuring a unique 5,5,5-fused tricyclic spiroketal butyrolactone moiety and diterpenoid-derived long chain, were isolated from Dysoxylum hongkongense. Their structures were unambiguously assigned by a combination of spectroscopic data, chemical degradation, Xray crystallography, CD analysis, and total synthesis. The total syntheses of compounds 1-4 were effectively accomplished by a convergent strategy with the longest linear sequences of 12-14 steps and overall yields of 5.4- 9.6%. Notably, we exploited a bioinspired one-pot method to construct the key intermediate 14 from an easily made compound 12 by involving the cascade reactions of an elaborate Claisen rearrangement, deprotections, and a 5-exo-trig cyclization. The desired major epimer 14a was then transformed to the main building block 21. Assembly of 21 and the long chain vinyl iodide 7 was made by an NHK coupling reaction to furnish the framework of 1-4. Some of the hongkonoids and/or synthetic analogs showed significant to moderate inhibitory activities against NF-κB, 11β-HSD1, and sterol synthesis. The most active NF-κB inhibitor 34 exhibited distinct inhibition on the LPS-induced inflammatory responses in RAW 246.7 and primary BMDM cells. [ABSTRACT FROM AUTHOR]

The clinical translation of protein drugs that act intracellularly is limited by the absence of safe and efficient intracellular protein delivery vehicles. Here, pH-sensitive coiled-coil peptide-cross-linked hyaluronic acid nanogels (HA-cNGs) were designed and investigated for targeted intracellular protein delivery to CD44 overexpressing MCF-7 breast cancer cells. HA-cNGs were obtained with a small size of 176 nm from an equivalent mixture of hyaluronic acid conjugates with GY(EIAALEK)3GC (E3) and GY(KIAALKE)3GC (K3) peptides, respectively, at pH 7.4 by nanoprecipitation. Circular dichroism (CD) proved the formation of coiled-coil structures between E3 and K3 peptides at pH 7.4 while fast uncoiling at pH 5.0. HA-cNGs showed facile loading of cytochrome C (CC) and greatly accelerated CC release under mild acidic conditions (18.4%, 76.8%, and 91.4% protein release in 24 h at pH 7.4, 6.0, and 5.0, respectively). Confocal microscopy and flow cytometry displayed efficient internalization of CC-loaded HA-cNGs and effective endosomal escape of CC in MCF-7 cancer cells. Remarkably, HA-cNGs loaded with saporin, a ribosome inactivating protein, exhibited significantly enhanced apoptotic activity to MCF-7 cells with a low IC50 of 12.2 nM. These coiled-coil peptide-cross-linked hyaluronic acid nanogels have appeared as a simple and multifunctional platform for efficient intracellular protein delivery. [ABSTRACT FROM AUTHOR]

The fundamental biogeochemical cycle of nitrogen includes cytochrome c nitrite reductase, which catalyzes the reduction of nitrite ions to ammonium with eight protons and six electrons (NO2- + 8H+ + 6e- → NH4+ + 2H2O). This reaction has motivated researchers to explore the reduction of nitrite. Although a number of electrochemical reductions of NO2- have been reported, the synthetic nitrite reduction reaction remains limited. To the best of our knowledge, formation of ammonia has not been reported. We report a three-step nitrite reduction cycle on a dinuclear ruthenium platform {(TpRu)2(μ-pz)} (Tp = HB(pyrazol-1-yl)3), producing ammonia. The cycle comprises conversion of a nitrito ligand to a NO ligand using 2H+ and e-, subsequent reduction of the NO ligand to a nitrido and a H2O ligand by consumption of 2H+ and 5e-, and recovery of the parent nitrito ligand. Moreover, release of ammonia was detected. [ABSTRACT FROM AUTHOR]

Hydrazide group (-C(O)-NH-NH2) is considered as a structural alert in the drug discovery process because the biotransformation chemistry of this group leads to the generation of toxic radical intermediates. The most important antitubercular drug isoniazid (INH) carries the hydrazide group. The toxicity of INH has been attributed to the protein adduct formation involving isonicotinoyl radical. However, the structures of reactive metabolites (RMs) and metabolite intermediate complexes (MICs), as well as the reaction mechanism for the formation and fate of RMs/MICs, have not been established. This report provides a detailed account of the biotransformation of INH by cytochromes using quantum chemical (QC) methods. Two cycles of cytochrome catalysis are involved in the formation of the most important RM, isonicotinoyl radical. The first cycle requires ~11 kcal/mol barrier on the oxidation pathway involving the formation of the RM isonicotinoyldiazene. The second cycle involves a barrier of ~7 kcal/mol for the activation of the diazene intermediate leading to isonicotinic acid via three reaction steps: (i) N-H bond activation, (ii) loss of N2 molecule, and (iii) rebound of isonicotinoyl radical. The RMs on the pathway (diazene, isonicotinoyl radical, N-hydroxy diazene) can react with the porphyrin ring/the amino acids of the cytochrome leading to many MICs (at least nine varieties), which can cause mechanism based inhibition and drug-drug interactions. This QC, molecular docking, and QM/MM study explored all the above reaction pathways and established the 3D structures of the RMs and MICs. [ABSTRACT FROM AUTHOR]

In addition to serving as respiratory electron shuttle, ferricytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by H2O2. This peroxidase function plays a key role during apoptosis. Typical peroxidases have a five-coordinate heme with a vacant distal coordination site that permits the iron center to interact with H2O2. In contrast, native cyt c is six-coordinate, as the distal coordination site is occupied by Met80. It thus seems counterintuitive that native cyt c would exhibit peroxidase activity. The current work scrutinizes the origin of this structure-function mismatch. Cyt c-catalyzed peroxidase reactions show an initial lag phase that is consistent with the in situ conversion of a precatalyst to an active peroxidase. Using mass spectrometry, we demonstrate the occurrence of cyt c self-oxidation in the presence of H2O2. The newly generated oxidized proteoforms are shown to possess significantly enhanced peroxidase activity. H2O2-induced modifications commence with oxidation of Tyr67, followed by permanent displacement of Met80 from the heme iron. The actual peroxidase activation step corresponds to subsequent side chain carbonylation, likely at Lys72/73. The Tyr67-oxidized/carbonylated protein has a vacant distal ligation site, and it represents the true peroxidase-active structure of cyt c. Subsequent self-oxidation eventually causes deactivation. It appears that this is the first report that identifies H2O2-induced covalent modifications as an essential component for the peroxidase activity of "native" cyt c. [ABSTRACT FROM AUTHOR]

Globular proteins, such as cytochrome c (cyt c), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt c was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the -5 to -10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt c major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity. [ABSTRACT FROM AUTHOR]

Ionic liquids (ILs) have now been acknowledged as reaction media for biotransformations. The first three examples were reported in this field in 2000, and since then, numerous applications have been reported for biocatalytic reactions using ILs. Two topics using ILs for enzymatic reactions have been reviewed from the standpoint of biocatalyst mediating organic synthesis; the first is "Biocatalysis in Ionic Liquids" which includes various types of biocatalytic reactions in ILs (section 2): (1) recent examples of lipase-mediated reactions using ILs as reaction media for biodiesel oil production and for sugar ester production, (2) oxidase-catalyzed reactions in ILs, (3) laccase-catalyzed reactions, (4) peroxidase-catalyzed reactions, (4) cytochrome-mediated reactions, (5) microbe-mediated hydrations, (6) protease-catalyzed reactions, (8) whole cell mediated asymmetric reduction of ketones, (10) acylase-catalyzed reactions, (11) glycosylation or cellulase-mediated hydrolysis of polysaccharides, (12) hydroxynitrile lyase-catalyzed reaction, (13) fluorinase or haloalkane dehydrogenase-catalyzed reaction, (14) luciferase-catalyzed reactions, and (15) biocatalytic promiscuity of enzymatic reactions for organic synthesis using ILs. The second is "Enzymes Activated by Ionic Liquids for Organic Synthesis", particularly describing the finding story of activation of lipases by the coating with a PEG-substituted IL (section 3). The author's opinion toward "Future Perspectives of Using ILs for Enzymatic Reactions" has also been discussed in section 4. [ABSTRACT FROM AUTHOR]

The homodimeric bc1 protein complex is embedded in membranes of mitochondria and photosynthetic bacteria, where it transports protons across the membrane to maintain an electrostatic potential used to drive ATP synthesis as part of the respiratory or photosynthetic pathways. The reaction cycle of the bc1 complex is driven by series of redox processes involving substrate molecules from the membrane, but occasional side reactions between an intermediate semiquinone substrate and molecular oxygen are suspected to be a source of toxic superoxide, which is believed to be a factor in aging. The present investigation employs molecular dynamics simulations to study the effect of mutations in the Qo binding sites of the bc1 complex on the ability of oxygen molecules to migrate to and bind at various locations within the complex. It is found that the mutations strongly affect the ability of oxygen to bind at the Qo sites, and moreover, different behavior of the two monomers of the bc1 complex is observed. The conformational differences at the Qo sites of the two monomers are studied in detail and discussed. The anionic form of semiquinone was identified as leading to the greatest opportunity for side reactions with oxygen. [ABSTRACT FROM AUTHOR]

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids. [ABSTRACT FROM AUTHOR]

The decaheme cytochrome MtrC from Shewanella oneidensis MR-1 immobilized on an ITO electrode displays unprecedented H2O2 reduction activity. Although MtrC showed lower peroxidase activity in solution compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. A hierarchical ITO electrode enabled optimal immobilization of MtrC and a high current density of 1 mA cm-2 at 0.4 V vs SHE could be obtained at pH 6.5 (Eonset = 0.72 V). UV-visible and Resonance Raman spectroelectrochemical studies suggest the formation of a high valent iron-oxo species as the catalytic intermediate. Our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish MtrC as a new benchmark in biotechnological H2O2 reduction with scope for applications in fuel cells and biosensors. [ABSTRACT FROM AUTHOR]

The cytochrome bc1 complex is the third protein complex in the electron transport chain of mitochondria or photosynthetic bacteria, and it serves to create an electrochemical gradient across a cellular membrane, which is used to drive ATP synthesis. The purpose of this study is to investigate interactions involving an occasionally trapped oxygen molecule (O2) at the so-called Qo site of the bc1 complex, which is one of the central active sites of the protein complex, where redox reactions are expected to occur. The investigation focuses on revealing the possibility of the oxygen molecule to influence the normal operation of the bc1 complex and acquire an extra electron, thus becoming superoxide, a biologically toxic free radical. The process is modeled by applying quantum chemical calculations to previously performed classical molecular dynamics simulations. Investigations reveal several spontaneous charge transfer modes from amino acid residues and cofactors at the Qo-site to the trapped O2 molecule. [ABSTRACT FROM AUTHOR]

Many adverse drug reactions are thought to be caused by electrophilically reactive drug metabolites that conjugate to nucleophilic sites within DNA and proteins, causing cancer or toxic immune responses. Quinone species, including quinone-imines, quinone-methides, and imine-methides, are electrophilic Michael acceptors that are often highly reactive and comprise over 40% of all known reactive metabolites. Quinone metabolites are created by cytochromes P450 and peroxidases. For example, cytochromes P450 oxidize acetaminophen to N-acetyl-p-benzoquinone imine, which is electrophilically reactive and covalently binds to nucleophilic sites within proteins. This reactive quinone metabolite elicits a toxic immune response when acetaminophen exceeds a safe dose. Using a deep learning approach, this study reports the first published method for predicting quinone formation: the formation of a quinone species by metabolic oxidation. We model both one- and two-step quinone formation, enabling accurate quinone formation predictions in nonobvious cases. We predict atom pairs that form quinones with an AUC accuracy of 97.6%, and we identify molecules that form quinones with 88.2% AUC. By modeling the formation of quinones, one of the most common types of reactive metabolites, our method provides a rapid screening tool for a key drug toxicity risk. The XenoSite quinone formation model is available at http://swami.wustl.edu/xenosite/p/quinone. [ABSTRACT FROM AUTHOR]

The intramembrane cytochrome bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus and the cytochrome b6f complex, which functions in oxygenic photosynthesis, utilize two pairs of b-hemes in a symmetric dimer to accomplish proton-coupled electron transfer. The transmembrane electron transfer pathway in each complex was identified through the novel use of heme Soret band excitonic circular dichroism (CD) spectra, for which the responsible heme-heme interactions were determined from crystal structures. Kinetics of heme reduction and CD amplitude change were measured simultaneously. For bc1, in which the redox potentials of the transmembrane heme pair are separated by 160 mV, heme reduction occurs preferentially to the higher-potential intermonomer heme pair on the electronegative (n) side of the complex. This contrasts with the b6f complex, where the redox potential difference between transmembrane intramonomer p- and n-side hemes is substantially smaller and the n-p pair is preferentially reduced. Limits on the dielectric constant between intramonomer hemes were calculated from the interheme distance and the redox potential difference, ΔEm. The difference in preferred reduction pathway is a consequence of the larger ΔEm between n- and p-side hemes in bc1, which favors the reduction of n-side hemes and cannot be offset by decreased repulsive Coulombic interactions between intramonomer hemes. [ABSTRACT FROM AUTHOR]

The bc1 complex is a critical enzyme for the ATP production in photosynthesis and cellular respiration. Its biochemical function relies on the so-called Q-cycle, which is well established and operates via quinol substrates that bind inside the protein complex. Despite decades of research, the quinol-protein interaction, which initiates the Q-cycle, has not yet been completely described. Furthermore, the initial charge transfer reactions of the Q-cycle lack a physical description. The present investigation utilizes classical molecular dynamics simulations in tandem with quantum density functional theory calculations, to provide a complete and consistent quantitative description of the primary events that occur within the bc1 complex upon quinol binding. In particular, the electron and proton transfer reactions that trigger the Q-cycle in the bc1 complex from Rhodobacter capsulatus are studied. The coupled nature of these charge transfer reactions was revealed by obtaining the transition energy path connecting configurations of the Qo-site prior and after the transfers. The analysis of orbitals and partial charge distribution of the different states of the Qo-site has further supported the conclusion. Finally, key structural elements of the bc1 complex that trigger the charge transfer reactions were established, manifesting the importance of the environment in the process, which is furthermore evidenced by free energy calculations. [ABSTRACT FROM AUTHOR]

The aggregation behavior of 9, 36, and 112 nm hematite particles was studied in the presence of OmcA, a bacterial extracellular protein, in aqueous dispersions at pH 5.7 through time-resolved dynamic light scattering, electrophoretic mobility, and circular dichroism spectra, respectively. At low salt concentration, the attachment efficiencies of hematite particles in all sizes first increased, then decreased, and finally remained stable with the increase of OmcA concentration, indicating the dominant interparticle interaction changed along with the increase in the protein-to-particle ratio. Nevertheless, at high salt concentration, the attachment efficiencies of all hematite samples gradually decreased with increasing OmcA concentration, which can be attributed to increasing steric force. Additionally, the aggregation behavior of OmcA-hematite conjugates was more correlated to total particle-surface area than primary particle size. It was further established that OmcA could stabilize hematite nanoparticles more efficiently than bovine serum albumin (BSA), a model plasma protein, due to the higher affinity of OmcA to hematite surface. This study highlighted the effects of particle properties, solution conditions, and protein properties on the complicated aggregation behavior of protein-nanoparticle conjugates in aqueous environments. [ABSTRACT FROM AUTHOR]

A major goal in metalloprotein design is to build protein scaffolds from scratch that allow precise control over metal coordination. A particular challenge in this regard is the construction of allosteric systems in which metal coordination equilibria are coupled to other chemical events that take place elsewhere in the protein scaffold. We previously developed a metal-templated self-assembly strategy (MeTIR) to build supramolecular protein complexes with tailorable interfaces from monomeric building blocks. Here, using this strategy, we have incorporated multiple disulfide bonds into the interfaces of a Zn-templated cytochrome cb562 assembly in order to create mechanical strain on the quaternary structural level. Structural and biophysical analyses indicate that this strain leads to an allosteric system in which Zn2+ binding and dissociation are remotely coupled to the formation and breakage of a disulfide bond over a distance of >14 Å. The breakage of this strained bond upon Zn2+ dissociation occurs in the absence of any reductants, apparently through a hydrolytic mechanism that generates a sulfenic acid/thiol pair. [ABSTRACT FROM AUTHOR]

A family of triheme cytochromes from Geobacter sulfurreducens plays an important role in extracellular electron transfer. In addition to their role in electron transfer pathways, two members of this family (PpcA and PpcD) were also found to be able to couple e-/H+ transfer through the redox Bohr effect observed in the physiological pH range, a feature not observed for cytochromes PpcB and PpcE. In attempting to understand the molecular control of the redox Bohr effect in this family of cytochromes, which is highly homologous both in amino acid sequence and structures, it was observed that residue 6 is a conserved leucine in PpcA and PpcD, whereas in the other two characterized members (PpcB and PpcE) the equivalent residue is a phenylalanine. To determine the role of this residue located close to the redox Bohr center, we replaced Leu6 in PpcA with Phe and determined the redox properties of the mutant, as well as its solution structure in the fully reduced state. In contrast with the native form, the mutant PpcAL6F is not able to couple the e-/H+ pathway. We carried out the reverse mutation in PpcB and PpcE (i.e., replacing Phe6 in these two proteins by leucine) and the mutated proteins showed an increased redox Bohr effect. The results clearly establish the role of residue 6 in the control of the redox Bohr effect in this family of cytochromes, a feature that could enable the rational design of G. sulfurreducens strains that carry mutant cytochromes with an optimal redox Bohr effect that would be suitable for various biotechnological applications. [ABSTRACT FROM AUTHOR]

In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe2+CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states. [ABSTRACT FROM AUTHOR]

Flavins are known to enhance extracellular electron transfer (EET) in Shewanella oneidensis MR-1 bacteria, which reduce electron acceptors through outer-membrane (OM) cytochromes c. Free-shuttle and bound-redox cofactor mechanisms were proposed to explain this enhancement, but recent electrochemical reports favor a flavin-bound model, proposing two one-electron reductions of flavin, namely, oxidized (Ox) to semiquinone (Sq) and semiquinone to hydroquinone (Hq), at anodic and cathodic conditions, respectively. In this work, to provide a mechanistic understanding of riboflavin (RF) binding at the multiheme OM cytochrome OmcA, we explored binding configurations at hemes 2, 5, 7, and 10. Subsequently, on the basis of molecular dynamics (MD) simulations, binding free energies and redox potential shifts upon RF binding for the Ox/Sq and Sq/Hq reductions were analyzed. Our results demonstrated an upshift in the Ox/Sq and a downshift in the Sq/Hq redox potentials, consistent with a bound RF-OmcA model. Furthermore, binding free energy MD simulations indicated an RF binding preference at heme 7. MD simulations of the OmcA-MtrC complex interfacing at hemes 5 revealed a small interprotein redox potential difference with an electron transfer rate of 107-108/s. [ABSTRACT FROM AUTHOR]

Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ΔAmax/Ks values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra. [ABSTRACT FROM AUTHOR]

Glycopeptide antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding site on the X-domain and how the order of the cyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyBtei and OxyAtei. We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-linked peptide. On the basis of steady state kinetics of the OxyBtei-catalyzed reaction, we propose a model for P450 recruitment and peptide modification that involves continuous association/dissociation of the P450 enzymes with the NRPS, followed by specific recognition of the peptide cyclization state by the P450 (scanning). This leads to an induced conformational change that enhances the affinity of the enzyme/substrate complex and initiates catalysis; product release then occurs, with the product itself becoming the substrate for the second enzyme in the pathway. This model rationalizes our experimental findings for this complex enzyme cascade and provides insights into the orchestration of the sequential peptide tailoring reactions on the terminal NRPS module in GPA biosynthesis. [ABSTRACT FROM AUTHOR]

Metalloporphyrin containing proteins, such as cytochrome P450, play a key role in biological systems. The spectroscopic properties of metalloporphyrins have been a subject of intense interest and intense debate for over 50 years. Iron-porphyrins are usually believed to be nonfluorescent. Herein we report that, contrary to this belief, cytochrome P450 heme groups luminesce with enough intensity to be of use in the characterization of these enzymes. To confirm that the emission is from the heme, we destroyed the heme by titration with cumene hydroperoxide and measured the changes in emission upon titration with compounds known to bind to the distal face of the heme in two human cytochrome P450 enzymes, known as CYP3A4 and CYP2C9. The titration curves gave spectral dissociation constants that were not significantly different from those reported using the Soret UV/vis absorbance changes. We have tentatively assigned the broad luminescence at ~500 nm to a ¹ππ* → gs fluorescence and the structured luminescence above 600 nm to a ³ππ* → gs phosphorescence. These assignments are not associated with the Q-band, and are in violation of Kasha's rule. To illustrate the utility of the emission, we measured spectral dissociation constants for testosterone binding to P450 3A4 in bilayers formed on glass coverslips, a measurement that would be very difficult to make using absorption spectroscopy. Complementary experiments were carried out with water-soluble P450cam. [ABSTRACT FROM AUTHOR]

8-Epidiosbulbin E acetate (EEA), a furanoid, was unexpectedly found to be the most abundant diterpenoid lactone in certain varieties of Dioscorea bulbifera L. (DB), a traditional herbal medicine widely used in Asian nations. This herb has been reported to cause liver injury in humans and experimental animals. The occurrence of EEA in DB was dependent on its commercial source. The present study shows that EEA exhibits time- and dose-dependent liver injury in mice. Pretreatment with ketoconazole prevented the animals from developing EEA-induced liver injury, caused 7- and 13-fold increases in the plasma Cmax and AUC of EEA, and decreased urinary excretion of glutathione conjugates derived from EEA. Pretreatment with buthionine sulfoximine exacerbated EEA-induced hepatotoxicity. In order to define the role of EEA's furan moiety in EEA-induced hepatotoxicity, we synthesized tetrahydro-EEA by catalytic hydrogenation of the furan moiety. No liver injury was observed in the animals given the same doses of tetrahydro-EEA as those used with EAA. The results indicate that EEA itself does not appear to be hepatotoxic but that the electrophilic intermediate generated by the metabolic activation of the furan ring mediated by cytochromes P450 is responsible for EEA-induced liver injury. [ABSTRACT FROM AUTHOR]

Recent evidence has established the colocalization of amyloid-rich plaques and heme-rich deposits in the human cerebral cortex as a common postmortem feature in Alzheimer's disease (AD). The amyloid β (Aβ) peptides have been shown to bind heme, and the resultant heme-Aβ complexes can generate toxic partially reduced oxygen species (PROS) and exhibit peroxidase activity. The heme-Aβ active site exhibits a concentration-dependent equilibrium between a high-spin mono-His-bound species similar to a peroxidase-type active site and a bis-His-bound six-coordinate low-spin species similar to that of a cytochrome b type active site. The νFe-His (241 cm-1) vibration has been identified in the high-spin heme-Aβ active site by resonance Raman spectroscopy. The formation of the low-spin heme-Aβ species is promoted by the His14 and noncoordinating second-sphere Arg5 residues. The high-spin state produces more PROS than the low-spin species. Nonbiological constructs modeling different forms of Aβ (oligomers, fibrils, etc.) suggest that the detrimental high-spin state is likely to dominate under most physiological conditions. [ABSTRACT FROM AUTHOR]

The potential of a Transversal Modulation Ion Mobility Spectrometry (TMIMS) instrument for protein analysis applications has been evaluated. The Collision Cross Section (CCS) of cytochrome c measured with the TMIMS is in agreement with values reported in the literature. Additionally, it enables tandem IMS-IMS prefiltration in dry gas and in vapor doped gas. The chemical specificity of the different dopants enables interesting studies on the structure of proteins as CCS changed strongly depending on the specific dopant. Hexane produced an unexpectedly high CCS shift, which can be utilized to evaluate the exposure of hydrophobic parts of the protein. Alcohols produced higher shifts with a dual behavior: an increase in CCS due to vapor uptake at specific absorption sites, followed by a linear shift typical for unspecific and unstable vapor uptake. The molten globule +8 shows a very specific transition. Initially, its CCS follows the trend of the compact folded states, and then it rapidly increases to the levels of the unfolded states. This strong variation suggests that the +8 charge state undergoes a dopant-induced conformational change. Interestingly, more sterically demanding alcohols seem to unfold the protein more effectively also in the gas phase. This study shows the capabilities of the TMIMS device for protein analysis and how tandem IMS-IMS with dopants could provide better understanding of the conformational changes of proteins. [ABSTRACT FROM AUTHOR]

We employed a combination of fluorescence,visible circular dichroism,and absorption spectroscopy to study the conformational changes offerricytochrome cupon its binding to cardiolipin-containingsmall unilamellar vesicles. The measurements were performed as a functionof the cardiolipin concentration, the cardiolipin content of the liposomes,and the NaCl concentration of the solvent. The data were analyzedwith a novel model that combines a single binding step with a conformationalequilibrium between native-like and non-native-like proteins boundto the membrane surface. The equilibrium between the two conformations,which themselves are comprised of structurally slightly differentsubconformations, shifts to the more non-native-like conformationwith increasing cardiolipin concentration. For the binding isothermsdescribed in this paper, we explicitly considered the enthalpic andentropic contributions of molecular crowding to protein binding atlow lipid concentrations and high occupancy of the liposome surface.Increasing the CL content of liposomes increases the overall bindingaffinity but makes the conformational distribution much more susceptibleto the influence of sodium and chloride ions, which shifts the equilibriumtoward the more native-like state and directly inhibits binding, particularlyto liposomes with 100% cardiolipin content. Spectroscopic evidencefurther suggests that a fraction of the non-native conformers adoptsa pentacoordinated state similar to those obtained in class C peroxidases.On the basis of our results, we propose a hypothesis that describesthe balance between facilitating and impeding forces controlling theperoxidase activity of cytochrome cin the innermembrane space of mitochondria. [ABSTRACT FROM AUTHOR]

Cytochrome aa3-600 is a terminal oxidase in the electron transport W-T Vdq pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochromec when it reduces dioxygen to water. The enzyme J stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for 00 vo 2.0 3.0 4 0 v. catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made y the R70H mutant and have characterized the menasemiquinone radical by advanced X- v- R 70H and Q-band EPR The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a14N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e2qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nt. from the Arg70 side chain. Analysis of the 1H2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ, and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function. [ABSTRACT FROM AUTHOR]

Metal-respiring bacteria are microorganismscapable of oxidizingorganic pollutants present in wastewater and transferring the liberatedelectrons to an electrode. This ability has led to their applicationas catalysts in bioelectrochemical systems (BESs), a sustainable technologycoupling bioremediation to electricity production. Crucial for thefunctioning of these BESs is a complex protein architecture consistingof several surface-exposed multiheme proteins, called outer membranecytochromes, wiring the cell metabolism to the electrode. Althoughthe role of these proteins has been increasingly understood, littleis known about the protein–electrode interactions and theirimpact on the performance of BESs. In this study, we used surface-enhancedresonance Raman spectroscopy in combination with electrochemical techniquesto unravel the nature of the protein–electrode interactionfor the outer membrane cytochrome OmcB from Desulfuromonasacetoxidans(Dace). Comparing the spectroelectrochemicalproperties of OmcB bound directly to the electrode surface with thoseof the same protein embedded inside an electroactive biofilm, we haveshown that the surface-exposed cytochromes of Dacebiofilms are in direct contact with the electrode surface. Evenif direct binding causes protein denaturation, the biofilm possessesthe ability to minimize the extent of the damage maximizing the amountof cells in direct electrical communication with the electrode. [ABSTRACT FROM AUTHOR]

Domain swapping that contributes to the stability of biologically crucial multisubunit complexes has been implicated in protein oligomerization. In the case of membrane protein assemblies, domain swapping of the iron--sulfur protein (ISP) subunit occurs in the hetero-oligomeric cytochrome b6f and bc1, complexes, which are organized as symmetric dimers that generate the transmembrane proton electrochemical gradient utilized for ATP synthesis. In these complexes, the ISP C-terminal predominantly β-sheet extrinsic domain containing the redox-active [2Fe-2S] cluster resides on the electrochemically positive side of each monomer in the dimeric complex. This domain is bound to the membrane sector of the complex through an N-terminal transmembrane α-helix that is "swapped' to the other monomer of the complex where it spans the complex and the membrane. Detailed analysis of the function and structure of the b6f complex isolated from the cyanobacterium Fremyella diplosiphon SF33 shows that the domain-swapped ISP structure is necessary for function but is not necessarily essential for maintenance of the dimeric structure of the complex. On the basis of crystal structures of the cytochrome complex, the stability of the cytochrome dimer is attributed to specific intermonomer protein--protein and protein--lipid hydrophobic interactions. The geometry of the domain-swapped ISP structure is proposed to be a consequence of the requirement that the anchoring helix of the ISP not perturb the heme organization or quinone channel in the conserved core of each monomer. [ABSTRACT FROM AUTHOR]

Photosyntheticorganisms use light to convert the inorganic matterin organic one. Photosynthetic process consists on several steps,and one of them involves plastoquinone (PQ) that acts as electronand proton shuttle between photosystem II and cytochrome. We preparedmembranes that mimic the characteristics and composition of naturalphotosynthetic membranes and we characterized them using several techniquesin order to obtain both the PQ molecules disposition in the membraneand their electrochemical behavior. The selected lipid was monogalactosyldiacylglycerol(MGDG) that represents the 50% of the lipid content of the thylakoidmembrane. Both MGDG and PQ, and the MGDG:PQ mixtures have been studiedusing surface pressure–area isotherms and the presence of PQalters the physical state and compactness of the MGDG matrix. Langmuir–Blodgett(LB) films have been obtained by transferring a monolayer that mimicshalf of the bilayer of a biological membrane. The AFM topographicalcharacterization of the monolayers on mica indicates the presenceof differentiated domains, corresponding to different physical stateslinked to the influence of the PQ content. Moreover, the electrochemicalbehavior of the monolayers has been studied when transferred on ITO,observing one main electrochemical process that is due to the divingposition of PQ molecules in the lipid matrix. [ABSTRACT FROM AUTHOR]

Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol ( m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Z) and the charge state with maximum intensity (Z) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Amyloidspecies with various morphologies have been found for differentproteins and disease systems. In this article, we aim to ask if thesemorphologies are unique to a particular protein or if they convertfrom one to another. Using a heme protein containing iron as the transition-metalactivator of aggregation and a negatively charged surfactant, partialunfolding of the protein and its aggregation have been induced. Inthe pathway of aggregation, we have observed the formation of severalmorphological structures of a single protein, which were visualizeddirectly using atomic force microscopy (AFM). These structures havebeen found to appear and disappear with time, and their formationcould be monitored under normal buffer conditions and at room temperaturewithout requiring any sophisticated chemical or biological methodologies.In addition, we have observed the formation of honeycomb-shaped morphology,which may serve as an intermediate. These amyloid-based nanostructuresmay have the potential to be explored in therapeutics delivery andother biomedical applications. [ABSTRACT FROM AUTHOR]

The article focuses on a study related to cellular respiration and perspective of protein pumping in the same. Topics discussed include coupling mechanism of respiratory electron transfer to the synthesis of adenosine triphosphate from adenosine diphosphate, generation of electrochemical proton gradient through oxidoreduction reactions of the respiratory chain, and arrangement of cytochromes in photosynthetic reaction center.

The article focuses on the reaction mechanism of bovine heart Cytochrome c Oxidase (CcO). Topics covered include the purification and crystallization of bovine heart CcO, how CcO lowers molecular oxygen (O2) in a reaction that is linked with proton pumping, structure and content of the metal cofactors of bovine heart CcO, and the structure and content of lipids.

Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis o f these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization o f the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism o f interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V. [ABSTRACT FROM AUTHOR]

We have developed an aptameric nanosensor for fluorescence activation imaging of cytochrome c (Cyt c). Fluorescence imaging tools that enable visualization of key molecular players in apoptotic signaling are essential for cell biology and clinical theranostics. Cyt c is a major mediator in cell apoptosis. However, fluorescence imaging tools allowing direct visualization of Cyt c translocation in living cells have currently not been realized. We report for the first time the realization of a nanosensor tool that enables direct fluorescence activation imaging of Cyt c released from mitochondria in cell apoptosis. This strategy relies on spatially selective cytosolic delivery of a nanosensor constructed by assembly of a fluorophore-tagged DNA aptamer on PEGylated graphene nanosheets. The cytosolic release of Cyt c is able to dissociate the aptamer from graphene and trigger an activated fluorescence signal. The nanosensor is shown to exhibit high sensitivity and selectivity, rapid response, large signal-to-background ratio for in vitro, and intracellular detection of Cyt c. It also enables real-time visualization of the Cyt c release kinetics and direct identification of the regulators for apoptosis. The developed nanosensor may provide a very valuable tool for apoptotic studies and catalyze the fundamental interrogations of Cyt c-mediated biology. [ABSTRACT FROM AUTHOR]

A pulsed hydrogen/deuterium exchange (HDX) method has been developed for rapid monitoring of the exchange kinetics of protein ions with DO a few milliseconds after electrospray ionization (ESI). The stepwise gradual evolution of HDX of multiply charged protein ions was monitored using the pulsed HDX mass spectrometry technique. Upon introducing a very short pulse of DO (in the μs to ms time scale) into the linear ion trap (LIT) of a time-of-flight (TOF) mass spectrometer, bimodal distributions were detected for the ions of cytochrome c and ubiquitin. Mechanistic details of HDX reactions for ubiquitin and cytochrome c in the gas phase were uncovered and the structural transitions were followed by analyzing the kinetics of HDX. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The increase of conductivity of electrolytesfavors the currentproduction in microbial fuel cells (MFCs). Adaptation of cell culturesto higher ionic strength is a promising strategy to increase electricityproduction. The bacterium Geobacter sulfurreducensis considered a leading candidate for MFCs. Therefore, it is importantto evaluate the impact of the ionic strength on the functional propertiesof key periplasmic proteins that warrants electron transfer to cellexterior. The effect of the ionic strength on the functional propertiesof triheme cytochrome PpcA, the most abundant periplasmic cytochromein G. sulfurreducens, was investigatedby NMR and potentiometric methods. The redox properties of heme IVare the most affected ones. Chemical shift perturbation measurementson the backbone NMR signals, at increasing ionic strength, also showedthat the region close to heme IV is the most affected due to the largenumber of positively charged residues, which confer a highly positiveelectrostatic surface around this heme. The shielding of these positivecharges at high ionic strength explain the observed decrease in thereduction potential of heme IV and shows that PpcA was designed tomaintain its functional mechanistic features even at high ionic strength. [ABSTRACT FROM AUTHOR]

We report here that photoinitiated electron flow involving a metal-substituted (M = Mg, Zn) myoglobin (Mb) and its physiological partner protein, cytochrome b5 (cyt b5) can be “symmetrized”: the [Mb:cyt b5] complex stabilized by three D/E → K mutations on Mb (D44K/D60K/E85K, denoted MMb) exhibits both oxidative and reductive ET quenching of both the singlet and triplet photoexcited MMb states, the direction of flow being determined by the oxidation state of the cyt b5 partner. The first-excited singlet state of MMb (1MMb) undergoes ns-time scale reductive ET quenching by Fe2+2+cyt b5 as well as ns-time scale oxidative ET quenching by Fe3+cyt b5, both processes involving an ensemble of structures that do not interconvert on this time scale. Despite a large disparity in driving force favoring photooxidation of 1MMb relative to photoreduction (δ(−ΔG0) ≈ 0.4 eV, M = Mg; ≈ 0.2 eV, M = Zn), for each M the average rate constants for the two reactions are the same within error, 1kf > 108 s–1. This surprising observation is explained by considering the driving-force dependence of the Franck–Condon factor in the Marcus equation. The triplet state of the myoglobin (3MMb) created by intersystem crossing from 1MMb likewise undergoes reductive ET quenching by Fe2+cyt b5 as well as oxidative ET quenching by Fe3+cyt b5. As with singlet ET, the rate constants for oxidative ET quenching and reductive ET quenching on the triplet time scale are the same within error, 3kf ≈ 105 s–1, but here the equivalence is attributable to gating by intracomplex conversion among a conformational ensemble. [ABSTRACT FROM AUTHOR]

The reaction center (RC) complex of the green sulfur bacterium Chlorobaculum tepidum is composed of the Fenna-Matthews-Olson antenna protein (FMO) and the reaction center core (RCC) complex. The RCC complex has four subunits: PscA, PscB, PscC, and PscD. We studied the FMO/RCC complex by chemically cross-linking the purified sample followed by biochemical and spectroscopic analysis. Blue-native gels showed that there were two types of FMO/RCC complexes, which are consistent with complexes with one copy of FMO per RCC and two copies of FMO per RCC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples after cross-linking showed that all five subunits of the RC can be linked by three different cross-linkers: bissulfosuccinimidyl suberate, disuccinimidyl suberate, and 3,3-dithiobis-sulfosuccinimidyl propionate. The interaction sites of the cross-linked complex were also studied using liquid chromatography coupled to tandem mass spectrometry. The results indicated that FMO, PscB, PscD, and part of PscA are exposed on the cytoplasmic side of the membrane. PscD helps stabilize FMO to the reaction center and may facilitate transfer of the electron from the RC to ferredoxin. The soluble domain of the heme-containing cytochrome subunit PscC and part of the core subunit PscA are located on the periplasmic side of the membrane. There is a close relationship between the periplasmic portions of PscA and PscC, which is needed for the efficient transfer of the electron between PscC and P840. [ABSTRACT FROM AUTHOR]

Cytochrome aa3 from Paracoccus denitrificans and cytochrome ba3 from Thermus thermophilus, two distinct members of the heme—copper oxidase superfamily, were immobilized on electrodes modified with gold nanoparticles. This procedure allowed us to achieve direct electron transfer between the enzyme and the gold nanoparticles and to obtain evidence for different electrocatalytic properties of the two enzymes. The pH dependence and thermostability reveal that the enzymes are highly adapted to their native environments. These results suggest that evolution resulted in different solutions to the common problem of electron transfer to oxygen. [ABSTRACT FROM AUTHOR]

FMO enzymes (FMOs) play a key rolein the processes of detoxificationand/or bioactivation of specific pharmaceuticals and xenobiotics bearingnucleophilic centers. The N-oxide and S-oxide metabolites produced by FMOs are often active metabolites.The FMOs are more active than cytochromes in the brain and work intandem with CYP3A4 in the liver. FMOs might reduce the risk of phospholipidosisof CAD-like drugs, although some FMOs metabolites seem to be neurotoxicand hepatotoxic. However, in silico methods for FMO metabolism predictionare not yet available. This paper reports, for the first time, a substrate-specificityand catalytic-activity model for FMO3, the most relevant isoform ofthe FMOs in humans. The application of this model to a series of compoundswith unknown FMO metabolism is also reported. The model has also beenvery useful to design compounds with optimal clearance and in findingerroneous literature data, particularly cases in which substanceshave been reported to be FMO3 substrates when, in reality, the experimentallyvalidated in silico model correctly predicts that they are not. [ABSTRACT FROM AUTHOR]

Thestaggered cross decaheme configuration of electron transfercofactors in the outer-membrane cytochrome MtrF serves as a prototypefor conformationally gated multiheme electron transport. Derived fromthe bacterium Shewanella oneidensis, the staggeredcross configuration reveals intersecting c-type octaheme and tetraheme“wires” containing thermodynamic “hills”and “valleys” (Proc. Natl. Acad. Sci. U. S.A.2014, 11, 611–616),suggesting that the protein structure may include a dynamical mechanismfor conductance and pathway switching depending on enzymatic functionalneed. Here, we applied classical molecular and statistical mechanicscalculations of large-amplitude protein dynamics in MtrF, to addressits potential to modulate pathway conductance, including assessmentof the effect of the total charge state. Explicit solvent moleculardynamics simulations of fully oxidized and fully reduced MtrF showedthat the slowest mode of collective decaheme motion is 90% similarbetween the oxidized and reduced states and consists primarily ofinterheme separation with minor rotational contributions. The frequencyof this motion is 1.7 × 107s–1,both for fully oxidized and fully reduced MtrF, slower than the downhillelectron transfer rates between stacked heme pairs at the octahemetermini and faster than the electron transfer rates between parallelhemes in the tetraheme chain. This implies that MtrF uses slow conformationalfluctuations to modulate electron flow along the octaheme pathway,apparently for the purpose of increasing the residence time of electronson lowest potential hemes 4 and 9. This apparent gating mechanismshould increase the success rate of electron transfer from MtrF tolow potential environmental acceptors via these two solvent-exposedhemes. [ABSTRACT FROM AUTHOR]

Perturbed matrix method calculationsare performed on a dihemecytochrome c(DHC) protein, in order to assign previouslyexperimentally detemined reduction potentials (E0) to their corresponding heme groups. Very good agreementbetween calculated values to experimental data prove that the presentapproach can be used as a predictive tool of redox thermodynamic propertiesof multicenter redox proteins in the absence of experimental data,or in synergy with state-of-the art spectroscopic and electrochemicalapproaches to obtain a detailed picture of electron transfer processeswithin these complex systems. [ABSTRACT FROM AUTHOR]

Thecytochrome b6fcomplex,a member of the cytochrome bcfamily thatmediates energy transduction in photosynthetic and respiratory membranes,is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membraneelectron transfer, quinone oxidation–reduction, and generationof a proton electrochemical potential. Analysis of electron storagein this pathway, utilizing simultaneous measurement of heme reduction,and of circular dichroism (CD) spectra, to assay heme–hemeinteractions, implies a heterogeneous distribution of the dielectricconstants that mediate electrostatic interactions between the fourhemes in the complex. Crystallographic information was used to determinethe identity of the interacting hemes. The Soret band CD signal isdominated by excitonic interaction between the intramonomer b-hemes, bnand bp, on the electrochemically negative and positive sidesof the complex. Kinetic data imply that the most probable pathwayfor transfer of the two electrons needed for quinone oxidation–reductionutilizes this intramonomer heme pair, contradicting the expectationbased on heme redox potentials and thermodynamics, that the two higherpotential hemes bnon different monomerswould be preferentially reduced. Energetically preferred intramonomerelectron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectricconstants. Relative to the medium separating the two higher potentialhemes bn, a relatively large dielectricconstant must exist between the intramonomer b-hemes,allowing a smaller electrostatic repulsion between the reduced hemes.Heterogeneity of dielectric constants is an additional structure–functionparameter of membrane protein complexes. [ABSTRACT FROM AUTHOR]

Pyrrolizidinealkaloids (PAs) are natural toxins widely distributedin plants. The toxic potencies of different PAs vary significantly.PAs are mono- or diesters of necine acids with a necine base. On thebasis of the necine bases, PAs are classified into three types: retronecine-type,otonecine-type, and platynecine-type. Hepatotoxic PAs contain an unsaturatednecine base. PAs exert hepatotoxicity through metabolic activationby hepatic cytochromes P450s (CYPs) to generate reactive intermediateswhich form pyrrole–protein adducts. These adducts provide amechanism-based biomarker to assess PA toxicity. In the present study,metabolic activation of 12 PAs from three structural types was investigatedfirst in mice to demonstrate significant variations in hepatic metabolicactivation of different PAs. Subsequently, the structural and enzymaticfactors affecting metabolic activation of these PAs were further investigatedby using human liver microsomes and recombinant human CYPs. Pyrrole–proteinadducts were detected in the liver and blood of mice and the in vitrosystems treated with toxic retronecine-type andotonecine-type PAs having unsaturated necine bases but not with aplatynecine-type PA containing a saturated necine base. Retronecine-typePAs produced more pyrrole–protein adducts than otonecine-typePAs with similar necine acids, demonstrating that the structure ofnecine base affected PA toxic potency. Among retronecine-type PAs,open-ring diesters generated the highest amount of pyrrole–proteinadducts, followed by macrocyclic diesters, while monoesters producedthe least. Only CYP3A4 and CYP3A5 activated otonecine-type PAs, whileall 10 CYPs studied showed the ability to activate retronecine-typePAs. Moreover, the contribution of major CYPs involved also variedsignificantly among retronecine-type PAs. In conclusion, our findingsprovide a scientific basis for predicting the toxicities of individualPAs in biological systems based on PA structural features and on thepattern of expression and the selectivity of the CYP isoforms present. [ABSTRACT FROM AUTHOR]

Fully electroactive multilayer architecturescombining the redoxprotein cytochrome cand the enzyme laccase by theuse of silica nanoparticles as artificial matrix have been constructedon gold electrodes capable of direct dioxygen reduction. Laccase form Trametes versicolorand cytochrome cfrom horse heart were electrostatically coimmobilized by alternatedeposition with interlayers of silica nanoparticles in a multilayerfashion. The layer formation has been monitored by quartz crystalmicrobalance. The electrochemical properties and performance of thenanobiomolecular entities were investigated by cyclic voltammetry,indicating, that a multistep electron transfer cascade, from the electrodevia cytochrome cin the layered system toward theenzyme laccase, and here to molecular dioxygen was achieved. The responseof the novel architecture is based on direct electron exchange betweenimmobilized proteins and can be tuned by the assembly process. [ABSTRACT FROM AUTHOR]

The article presents a study on metalloproteins with cytochrome, iron-sulfur and copper redox centers. It mentions the presence of the hemoprotein in the inner mitochondrial membrane of eukaryotic organisms as well as in Gram-positive and negative bacteria. An overview of the redox attributes of designed 6-coordinate heme proteins which imitate electron transfer (ET) cytochromes is also provided.