Faltung, Assemblierung und Stabilisierung des Transmembranproteins Cytochrom b 6

In dieser Arbeit wurde der Beitrag der interhelikalen Loops zur Faltung, Assemblierung und Stabilität des kofaktortragenden Transmembranproteins Cytochrom b6 in vitro untersucht. Cytochrom b6 ist aus vier Transmembranhelices aufgebaut, die über drei Loops miteinander verbunden sind. Die beiden nicht-kovalent gebundenen Kofaktoren werden spontan in der Häm-Bindespalte zwischen den zwei Cytochrom b6-Hälften gebunden. Die Ergebnisse zeigen, dass die Verlängerung oder Eliminierung des Loops, der die beiden Hälften verbindet, nicht die Faltung und Assemblierung des Proteins beeinflusst. Der Loop ist für eine räumliche Positionierung und Orientierung der Hälften während der Assemblierung nicht essentiell. Weiterhin scheint keiner der drei interhelikalen Loops für die Bindung der Kofaktoren notwendig zu sein. Die Cytochrom b6-Hälfte, bestehend aus den Helices A und B, besitzt eine Konformation, die stabil genug ist um Häm alleine zu binden. Ebenso zeigt Helix B alleine eine α-helikale Struktur und bindet ebenfalls Häm. In vivo wurden bislang keine Faktoren beschrieben, die an der Assemblierung beteiligt sind. Im Rahmen dieser Arbeit wurden strukturelle Merkmale des Häms identifiziert, welche die Spezifität der Häm-Bindung, wenigstens in vitro, ausmachen. Von großer Bedeutung ist dabei das zentrale Eisen-Ion, dessen Eliminierung oder Austausch die Häm-Bindung verhindert. Die Substituenten des Porphyrinrings scheinen hingegen für die Stabilität der Bindung notwendig zu sein.

Seawater carbonate chemistry, pigments and proteins during experiments with phytoplankton, 2010

In the high-nutrient, low-chlorophyll waters of the Gulf of Alaska, microcosm manipulation experiments were used to assess the effect of CO2 on growth and primary production under iron-limited and iron-replete conditions. As expected, iron had a strong effect on growth and photosynthesis. A modest and variable stimulation of growth and biomass production by CO2 (high CO2: 77-122 Pa; low CO2: 11-17 Pa) was observed under both iron-replete and iron-limited conditions, though near the limit of precision of our measurements in slow-growing low-iron experiments. Physiological acclimations responsible for the changes in growth were assessed. Under iron-limited conditions, growth stimulation at high CO2 appeared to result from an increase in photosynthetic efficiency, which we attribute to energy savings from down-regulation of the carbon concentrating mechanisms. In some cases, iron-rich photosynthetic proteins (PsbA, PsaC, and cytochrome b6) were down-regulated at elevated CO2in iron-limited controls. Under iron-replete conditions, there was an increase in growth rate and biomass at high CO2 in some experiments. This increase was unexpectedly supported by reductions in cellular carbon loss, most likely decreased respiration. We speculate that this effect may be due to acclimation to decreased pH rather than high CO2. The variability in responses to CO2 among experiments did not appear to be caused by differences in phytoplankton community structure and may reflect the sensitivity of the net response of phytoplankton to antagonistic effects of the several parameters that co-vary with CO2.

Tracking molecular evolution of photosynthesis by characterization of a major photosynthesis gene cluster from Heliobacillus mobilis

A DNA sequence has been obtained for a 35.6-kb genomic segment from Heliobacillus mobilis that contains a major cluster of photosynthesis genes. A total of 30 ORFs were identified, 20 of which encode enzymes for bacteriochlorophyll and carotenoid biosynthesis, reaction-center (RC) apoprotein, and cytochromes for cyclic electron transport. Donor side electron-transfer components to the RC include a putative RC-associated cytochrome c553 and a unique four-large-subunit cytochrome bc complex consisting of Rieske Fe-S protein (encoded by petC), cytochrome b6 (petB), subunit IV (petD), and a diheme cytochrome c (petX). Phylogenetic analysis of various photosynthesis gene products indicates a consistent grouping of oxygenic lineages that are distinct and descendent from anoxygenic lineages. In addition, H. mobilis was placed as the closest relative to cyanobacteria, which form a monophyletic origin to chloroplast-based photosynthetic lineages. The consensus of the photosynthesis gene trees also indicates that purple bacteria are the earliest emerging photosynthetic lineage. Our analysis also indicates that an ancient gene-duplication event giving rise to the paralogous bchI and bchD genes predates the divergence of all photosynthetic groups. In addition, our analysis of gene duplication of the photosystem I and photosystem II core polypeptides supports a “heterologous fusion model” for the origin and evolution of oxygenic photosynthesis.

Seawater carbonate chemistry and the photophysiology of Thalassiosira pseudonana (Bacillariophyceae) and Emiliania huxleyi (Haptophyta) in a laboratory experiment

Increasing anthropogenic carbon dioxide is causing changes to ocean chemistry, which will continue in a predictable manner. Dissolution of additional atmospheric carbon dioxide leads to increased concentrations of dissolved carbon dioxide and bicarbonate and decreased pH in ocean water. The concomitant effects on phytoplankton ecophysiology, leading potentially to changes in community structure, are now a focus of concern. Therefore, we grew the coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler and the diatom strains Thalassiosira pseudonana (Hust.) Hasle et Heimdal CCMP 1014 and T. pseudonana CCMP 1335 under low light in turbidostat photobioreactors bubbled with air containing 390 ppmv or 750 ppmv CO2. Increased pCO2 led to increased growth rates in all three strains. In addition, protein levels of RUBISCO increased in the coastal strains of both species, showing a larger capacity for CO2 assimilation at 750 ppmv CO2. With increased pCO2, both T. pseudonana strains displayed an increased susceptibility to PSII photoinactivation and, to compensate, an augmented capacity for PSII repair. Consequently, the cost of maintaining PSII function for the diatoms increased at increased pCO2. In E. huxleyi, PSII photoinactivation and the counter-acting repair, while both intrinsically larger than in T. pseudonana, did not change between the current and high-pCO2 treatments. The content of the photosynthetic electron transport intermediary cytochrome b6/f complex increased significantly in the diatoms under elevated pCO2, suggesting changes in electron transport function.

Cytochrome p450 isozyme protein verified in the skin of southern hemisphere humpback whales (megaptera novaeangliae) : implications for biochemical biomarker assessment

Large mysticete whales represent a unique challenge for chemical risk assessment. Few epidemiological investigations are possible due to the low incidence of adult stranding events. Similarly their often extreme life-history adaptations of prolonged migration and fasting challenge exposure assumptions. Molecular biomarkers offer the potential to complement information yielded through tissue chemical analysis, as well as providing evidence of a molecular response to chemical exposure. In this study we confirm the presence of cytochrome P450 reductase (CPR) and cytochrome P450 isoenzyme 1A1 (CYP1A1) in epidermal tissue of southern hemisphere humpback whales (Megaptera novaeangliae). The detection of CYP1A1 in the integument of the humpback whale affords the opportunity for further quantitative non-destructive investigations of enzyme activity as a function of chemical stress.

Electrochemical detection of dopamine and cytochrome c at a nanostructured gold electrode

A nanostructured gold surface consisting of closely packed outwardly growing spikes is investigated for the electrochemical detection of dopamine and cytochrome c. A significant electrocatalytic effect for the electrooxidation of both dopamine and ascorbic acid at the nanostructured electrode was found due to the presence of surface active sites which allowed the detection of dopamine in the presence of excess ascorbic acid to be achieved by differential pulse voltammetry. By simple modification with a layer of Nafion, the enhanced electrocatalytic properties of the nanostructured surface was maintained while increasing the selectivity of dopamine detection in the presence of interfering species such as excess ascorbic and uric acids. Also, upon modification of the nanostructured surface with a monolayer of cysteine, the electrochemical response of immobilised cytochrome c in two distinct conformations was observed. This opens up the possibility of using such a nanostructured surface for the characterisation of other biomolecules and in bio-electroanalytical applications.

Phenytoin metabolism by human cytochrome P450 : involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Enantioselective epoxidation of linolenic acid catalysed by cytochrome P450BM3 from Bacillus megaterium

Cytochrome P450BM3, from Bacillus megaterium, catalyses the epoxidation of linolenic acid 1 yielding 15,16-epoxyoctadeca-9,12-dienoic acid 2 with complete regio- and moderate enantio-selectivity (60% ee). The absolute configuration of the product is tentatively assigned as 15(R),16(S)-. The Michaelis–Menten parameters kcat and Km for the reaction were determined to be 3126 ± 226 min−1 and 24 ± 6 μM respectively.

Rapid identification of cytochrome P450cam variants by in vivo screening of active site libraries

The selection of cytochrome P450 enzymes from large variant libraries, and the subsequent use of these enzymes in preparative scale biotransformations, remains a formidable challenge due to the complexities of the associated electron transport systems. Here, a powerful approach for the generation and screening of P450cam libraries for new function is presented that is both flexible and robust. A targeted library was generated wherein only the P450cam active-site amino acids Y96 and F98 were fully randomized and biotransformations, using a novel P450cam whole-cell system, were screened by GC–MS for the hydroxylation of diphenylmethane. One in 50 of the reactions screened, including 16 different variants, produced 4-hydroxydiphenylmethane with up to 92% conversion observed in the case of the Y96A variant. These results demonstrate a primary example of the screening of P450cam libraries in a format that is compatible with extension to preparative scale reactions.

Delivery of nitric oxide for analysis of the function of cytochrome c'

On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas. We describe a number of techniques for steady-state UV/Vis spectrophotometry and pre-steady-state stopped-flow spectrophotometry analysis of cytochrome c'.

The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals : consequences for occupational and environmental medicine

The importance of the isoform CYP2E1 of the human cytochrome P-450 superfamily of enzymes for occupational and environmental medicine is derived from its unique substrate spectrum that includes a number of highly important high-production chemicals, such as aliphatic and aromatic hydrocarbons, solvents and industrial monomers (i.a. alkanes, alkenes, aromatic and halogenated hydrocarbons). Many polymorphic genes, such as CYP2E1, show considerable differences in allelic distribution between different human populations. The polymorphic nature of the human CYP2E1 gene is significant for inter-individual differences in toxicity of its substrates. Since the substrate spectrum of CYP2E1 includes many compounds of basic relevance to industrial toxicology, a rationale for metabolic interactions of different CYP2E1 substrates is provided. In-depth research into the inter-individual phenotypic differences of human CYP2E1 enzyme activities was enabled by the recognition that the 6-hydroxylation of the drug chlorzoxazone is mediated by CYP2E1. Studies on CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There are consistent ethnic differences in CYP2E1 enzyme expression, mostly demonstrated between European and Japanese populations, which point to a major impact of genetic factors. The most frequently studied genetic polymorphisms are the restriction fragment length polymorphisms PstI/RsaI (mutant allele: CYP2E1*5B) located in the 5′-flanking region of the gene, as well as the DraI polymorphism (mutant allele: CYP2E1*6) located in intron 6. These polymorphisms are partly related, as they form the common allele designated CYP2E1*5A. Striking inter-ethnic differences between Europeans and Asians appear with respect to the frequencies of the CYP2E1*5A allele (only approximately 5% of Europeans are heterozygous, but 37% of Asians are, whilst 6% of Asians are homozygous). Available studies indicate a wide variation in human CYP2E1 expression, which are very likely based on complex gene-environment interactions. Major inter-ethnic differences are apparent on the genotyping and the phenotyping levels. Selected cases are presented where inter-ethnic variations of CYP2E1 may provide likely explanations for unexplained findings concerning industrial chemicals that are CYP2E1 substrates. Possible consequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expressions of clinical symptoms of chemical toxicity, to results of biological monitoring of exposed workers, and to the interpretation of results of epidemiological or molecular-epidemiological studies.

Interactions of ingested food, beverage, and tobacco components involving human cytochrome P4501A2, 2A6, 2E1, and 3A4 enzymes

Human cytochrome P450 (P450) enzymes are involved in the oxidation of natural products found in foods, beverages, and tobacco products and their catalytic activities can also be modulated by components of the materials. The microsomal activation of aflatoxin B1 to the exo-3,9-epoxide is stimulated by flavone and 7,8-benzoflavone, and attenuated by the flavonoid naringenin, a major component of grapefruit. P4502E1 has been demonstrated to play a potentially major role in the activation of a number of very low-molecular weight cancer suspects, including ethyl carbamate (urethan), which is present in alcoholic beverages and particularly stone brandies. The enzyme (P4502E1) is also known to be inducible by ethanol. Tobacco contains a large number of potential carcinogens. In human liver microsomes a significant role for P4501A2 can be demonstrated in the activation of cigarette smoke condensate. Some of the genotoxicity may be due to arylamines. P4501A2 is also inhibited by components of crude cigarette smoke condensate. The tobacco-specific nitrosamines are activated by a number of P450 enzymes. Of those known to be present in human liver, P4501A2, 2A6, and 2E1 can activate these nitrosamines to genotoxic products.