A Pathfinder in High-Pressure Bioscience: In Memoriam of Gaston Hui Bon Hoa.

On 26 July 2020, our colleague and friend Dr [...].

Non-interventional study evaluating exposure to inhaled, low-dose methoxyflurane experienced by hospital emergency department personnel in France.

OBJECTIVES: Low-dose methoxyflurane is a non-opioid, inhaled analgesic administered via the Penthrox inhaler and was recently licensed in Europe for emergency relief of moderate-to-severe trauma-associated pain in conscious adults. This non-interventional study investigated occupational exposure to methoxyflurane in the hospital emergency department (ED) personnel during routine clinical practice. SETTING AND PARTICIPANTS: The study was conducted in two hospital ED triage rooms in France over a 2-week and 3-week period, respectively. Low-dose methoxyflurane analgesia was self-administered by patients via the inhaler under the supervision of nursing staff, per routine clinical practice. An organic vapour personal badge sampler was attached to the uniform of the nurses working in the treatment rooms throughout an 8-hour shift (total of 140 shifts during the study period). Seven-day ambient air monitoring of each treatment room was also performed. Methoxyflurane levels adsorbed in each badge sampler were measured by a central laboratory. The primary objective was to evaluate methoxyflurane exposure experience by the hospital ED nurses during an 8-hour shift. RESULTS: In 138 badge samplers, the median (range) concentration of methoxyflurane present following 8-hour nursing shifts was 0.017 (0.008, 0.736) ppm. This level was almost 900-fold lower than the previously reported 8-hour-derived maximal exposure level of 15 ppm; methoxyflurane exposure approaching this threshold was not documented in any badges. There was no correlation between the number of applications of low-dose methoxyflurane administered during a shift (range 0-5) and the vapour exposure measured on the personal badge samplers. CONCLUSIONS: This study indicates that nurses working in hospital EDs experience very low levels of occupational exposure to methoxyflurane vapour during routine clinical practice. These real-world data can provide reassurance to healthcare providers supervising patients receiving low-dose methoxyflurane analgesia via a Penthrox inhaler; further studies may inform exposure in other hospital ED settings.

Antimicrobial efficacy of copper-doped titanium surfaces for dental implants.

The aim of this in vitro study was to quantify the antibacterial effect of a copper-deposited titanium surface as a model for dental implants on the peri-implantitis-associated strain Porphyromonas gingivalis (DSM 20709). A spark-assisted anodization method in a combined deposition-anodization process was applied to deposit copper on discs made of titanium. This method allows the deposition of different concentrations of copper on the surface by varying the process time. Conventional culturing was used to investigate the adhesion of P. gingivalis onto the discs over 2, 4, and 6 h as well as to study the antibacterial effect of copper released in solution. The viability of the bacterial cells is strongly inhibited on copper-deposited discs and reaches a CFU reduction of 3 log-units after 6 h in comparison to the reference. The copper released in solution causes a reduction of 4 log-units after a 6 h incubation time. With a 6 h incubation time, the CFU count decreases with increasing copper concentrations on the disc (by 2% for the 1.3 µg/disc; 32% for the 5.6 µg/disc; and 34% for the 9.5 µg/disc). However, at a higher copper concentration of 17.7 µg/disc, after 6 h, the decrease in the CFU count is less pronounced than that observed in solution, where a further decrease is observed. In conclusion, copper-functionalized titanium significantly reduces the survival of adhered bacteria and decreases the viable bacterial count in the environment surrounding the titanium. Thus, the area surrounding implants is being protected by copper released from the surface, forming a "safe zone" for improved implant healing.

Electrochemically driven biocatalysis of the oxygenase domain of neuronal nitric oxide synthase in indium tin oxide nanoparticles/polyvinyl alcohol nanocomposite.

Nitric oxide synthase (NOS) plays a critical role in a number of key physiological and pathological processes. Investigation of electron-transfer reactions in NOS would contribute to a better understanding of the nitric oxide (NO) synthesis mechanism. Herein, we describe an electrochemically driven catalytic strategy, using a nanocomposite that consisted of the oxygenase domain of neuronal NOS (D290nNOSoxy), indium tin oxide (ITO) nanoparticles and polyvinyl alcohol (PVA). Fast direct electron transfer between electrodes and D290nNOSoxy was observed with the heterogeneous electron transfer rate constant (ket) of 154.8 ± 0.1s(-1) at the scan rate of 5 Vs(-1). Moreover, the substrate N(ω)-hydroxy-L-arginine (NHA) was used to prove the concept of electrochemically driven biocatalysis of D290nNOSoxy. In the presence of the oxygen cosubstrate and tetrahydrobiopterin (BH4) cofactor, the addition of NHA caused the decreases of both oxidation current at +0.1 V and reduction current at potentials ranging from -0.149 V to -0.549 V vs Ag/AgCl. Thereafter, a series of control experiments such as in the absence of BH4 or D290nNOSoxy were performed. All the results demonstrated that D290nNOSoxy biocatalysis was successfully driven by electrodes in the presence of BH4 and oxygen. This novel bioelectronic system showed potential for further investigation of NOS and biosensor applications.

Spectroscopic characterization of cytochrome P450 Compound I.

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom as Fe(IV)O is called Compound I (Cpd I). Cpd I is the intermediate species proposed to hydroxylate directly the inert carbon-hydrogen bonds of P450 substrates. In the natural reaction cycle of cytochrome P450 Cpd I has not yet been detected, presumably because it is very short-lived. A great variety of experimental approaches has been applied to produce Cpd I artificially aiming to characterize its electronic structure with spectroscopic techniques. In spite of these attempts, none of the spectroscopic studies of the last decades proved capable of univocally identifying the electronic state of P450 Cpd I. Very recently, however, Rittle and Green [9] have shown that Cpd I of CYP119, the thermophilic P450 from Sulfolobus acidocaldarius, is univocally a Fe(IV)O-porphyrin radical with the ferryl iron spin (S=1) antiferromagnetically coupled to the porphyrin radical spin (S'=1/2) yielding a S(tot)=1/2 ground state very similar to Cpd I of chloroperoxidase from Caldariomyces fumago. In this mini-review the efforts to characterize Cpd I of cytochrome P450 by spectroscopic methods are summarized.

The mystery of cytochrome P450 Compound I: a mini-review dedicated to Klaus Ruckpaul.

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom is called Compound I, which is presumably the intermediate species which hydroxylates inert carbon-hydrogen bonds of substrates. In this mini-review, the milestones in discovering Compound I of cytochrome P450 are summarized. It will be discussed what was known and suggested in the years before 1984, the year when Klaus Ruckpaul's first book about cytochrome P450 appeared, and compared with recent approaches and studies to catch and characterize this intermediate oxygen species in the reaction cycle of cytochrome P450. Although many studies have been undertaken before and after 1984 to characterize Compound I, its electronic structure and physicochemical properties are still a mystery. The conclusion from this review is that the knowledge about Compound I has significantly increased; however, we still ask the same questions. There is a need for improved experimental approaches, detection techniques, and theoretical simulations for future studies of cytochrome P450 Compound I. This mini-review is dedicated to Klaus Ruckpaul on the occasion of his 80th birthday.

Fourier transform infrared spectroscopy as a tool to study structural properties of cytochromes P450 (CYPs).

Cytochrome P450 proteins (CYPs) are a big class of heme proteins which are involved in various metabolic processes of living organisms. CYPs are the terminal catalytically active components of monooxygenase systems where the substrate binds and is hydroxylated. In order to be functionally competent, the protein structures of CYPs possess specific properties that must be explored in order to understand structure-function relationships and mechanistic aspects. Fourier transform infrared spectroscopy (FTIR) is one tool that is used to study these structural properties. The application of FTIR spectroscopy to the secondary structures of CYP proteins, protein unfolding, protein-protein interactions and the structure and dynamics of the CYP heme pocket is reviewed. A comparison with other thiolate heme proteins (nitric oxide synthase and chloroperoxidase) is also included.

About oxygen, cytochrome p450 and titanium: learning from Ron Estabrook.

We learned from Ron Estabrook to consider the complexity of the cytochrome P450 system and to appreciate insights coming from other fields. Two issues from different fields are comparatively discussed which both have formally in common to reflect the response of the human body on foreign compounds and materials. The former ones are environmental pollutants and drugs, while the latter are solid materials such as titanium, used for orthopedic implants. It will be reviewed that both show rich oxygen chemistry as catalysts and are involved in complex biochemical responses at different regulatory levels in foreign body reactions.

FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

Light-induced reduction of bovine adrenodoxin via the covalently bound ruthenium(II) bipyridyl complex: intramolecular electron transfer and crystal structure.

Bovine adrenodoxin (Adx) plays an important role in the electron-transfer process in the mitochondrial steroid hydroxylase system of the bovine adrenal cortex. Using electron paramagnetic resonance (EPR) spectroscopy, we showed that photoreduction of the [2Fe-2S] cluster of Adx via (4'-methyl-2,2'-bipyridine)bis(2,2'-bipyridine)ruthenium(II) [Ru(bpy)2(mbpy)] covalently attached to the protein surface can be used as a new approach to probe the "shuttle" hypothesis for the electron transfer by Adx. The 1.5 A resolution crystal structure of a 1:1 Ru(bpy)2(mbpy)-Adx(1-108) complex reveals the site of modification, Cys95, and allows to predict the possible intramolecular electron-transfer pathways within the complex. Photoreduction of uncoupled Adx, mutant Adx(1-108), and Ru(bpy)2(mbpy)-Adx(1-108) using safranin T as the mediating electron donor suggests that two electrons are transferred from the dye to Adx. The intramolecular photoreduction rate constant for the ruthenated Adx has been determined and is discussed according to the predicted pathways.

Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization.

Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450.

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.

Multiple molecular recognition mechanisms. Cytochrome P450--a case study.

Biomolecular recognition is complex. The balance between the different molecular properties that contribute to molecular recognition, such as shape, electrostatics, dynamics and entropy, varies from case to case. This, along with the extent of experimental characterization, influences the choice of appropriate computational approaches to study biomolecular interactions. Here, we present computational studies of cytochrome P450 enzymes and their interactions with small molecules and with other proteins. These interactions exemplify some of the diversity of molecular determinants of binding affinity and specificity observed for proteins and we discuss some of the challenges that they pose for molecular modelling and simulation.

Freeze-quenched iron-oxo intermediates in cytochromes P450.

Since the discovery of cytochromes P450 and their assignment to heme proteins a reactive iron-oxo intermediate as the hydroxylating species has been discussed. It is believed that the electronic structure of this intermediate corresponds to an iron(IV)-porphyrin-pi-cation radical system (Compound I). To trap this intermediate the reaction of P450 with oxidants (shunt pathway) has been used. The common approaches are stopped-flow experiments with UV-visible spectroscopic detection or rapid-mixing/freeze-quench studies with EPR and Mössbauer spectroscopic characterization of the trapped intermediate. Surprisingly, the two approaches seem to give conflicting results. While the stopped-flow data indicate the formation of a porphyrin-pi-cation radical, no such species is seen by EPR spectroscopy, although the Mössbauer data indicate iron(IV) for P450cam (CYP101) and P450BMP (CYP102). Instead, radicals on tyrosine and tryptophan residues are observed. These findings are reviewed and discussed with respect to intramolecular electron transfer from aromatic amino acids to a presumably transiently formed porphyrin-pi-cation radical.

Cytochrome P450 biosensors-a review.

Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation of their structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970s attempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electron transfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigation of the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of substrates important in environmental monitoring, pharmaceutical industry and clinical practice.

Thermostability and Ca2+ binding properties of wild type and heterologously expressed PsbO protein from cyanobacterial photosystem II.

Oxygenic photosynthesis takes place in the thylakoid membrane of cyanobacteria, algae, and higher plants. Initially light is absorbed by an oligomeric pigment-protein complex designated as photosystem II (PSII), which catalyzes light-induced water cleavage under release of molecular oxygen for the biosphere on our planet. The membrane-extrinsic manganese stabilizing protein (PsbO) is associated on the lumenal side of the thylakoids close to the redox-active (Mn)(4)Ca cluster at the catalytically active site of PSII. Recombinant PsbO from the thermophilic cyanobacterium Thermosynechococcus elongatus was expressed in Escherichia coli and spectroscopically characterized. The secondary structure of recombinant PsbO (recPsbO) was analyzed in the absence and presence of Ca(2+) using Fourier transform infrared spectroscopy (FTIR) and circular dichroism spectropolarimetry (CD). No significant structural changes could be observed when the PSII subunit was titrated with Ca(2+) in vitro. These findings are compared with data for spinach PsbO. Our results are discussed in the light of the recent 3D-structural analysis of the oxygen-evolving PSII and structural/thermodynamic differences between the two homologous proteins from thermophilic cyanobacteria and plants.

Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101).

Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida, has been characterized concerning thermal stability and structural dynamics. For the first time, Fourier transform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein. The secondary structure compositions were determined from the fitted amide I' spectral region, giving band populations at 10 degrees C for the perdeuterated protein of 22% between 1605 and 1624 cm(-1) (beta-sheets), 47% between 1633 and 1650 cm(-1) (alpha-helix (29%) plus unordered/3(10)-helix (18%)), and 28% between 1657 and 1677 cm(-1) (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm(-1) (beta-sheets), 52% between 1640 and 1658 cm(-1) (alpha-helix (41%) plus unordered/3(10)-helix (11%)), and 24% between 1665 and 1680 cm(-1) (turns). Thermal unfolding experiments revealed that perdeuterated P450cam was less stable than the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 degrees C for the perdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to the P450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of the ligand occurred with the same kinetics and rate constants k(on), 8.9 x 10(4) and 8.3 x 10(4) M(-1) s(-1) for the perdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutron crystallographic study to determine the specific hydration states and hydrogen-bonding networks at the active site. The analyses presented here show that perdeuterated P450cam is structurally similar to its hydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained from the neutron structure will be representative of the normal hydrogenated P450cam.

Substrate binding favors enhanced NO binding to P450cam.

Ferric cytochrome P450cam from Pseudomonas putida (P450cam) in buffer solution at physiological pH 7.4 reversibly binds NO to yield the nitrosyl complex P450cam(NO). The presence of 1R-camphor affects the dynamics of NO binding to P450cam and enhances the association and dissociation rate constants significantly. In the case of the substrate-free form of P450cam, subconformers are evident and the NO binding kinetics are much slower than in the presence of the substrate. The association and dissociation processes were investigated by both laser flash photolysis and stopped-flow techniques at ambient and high pressure. Large and positive values of S and V observed for NO binding to and release from the substrate-free P450cam complex are consistent with the operation of a limiting dissociative ligand substitution mechanism, where the lability of coordinated water dominates the reactivity of the iron(III)-heme center with NO. In contrast, NO binding to P450cam in the presence of camphor displays negative activation entropy and activation volume values that support a mechanism dominated by a bond formation process. Volume profiles for the binding of NO appear to be a valuable approach to explain the differences observed for P450cam in the absence and presence of the substrate and enable the clarification of the underlying reaction mechanisms at a molecular level. Changes in spin state of the iron center during the binding/release of NO contribute significantly to the observed volume effects. The results are discussed in terms of relevance for the biological function of cytochrome P450 and in context to other investigations of the related reactions between NO and imidazole- and thiolate-ligated iron(III) hemoproteins.

Spectroelectrochemistry of cytochrome P450cam.

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.