Studies on the interaction of NADPH cytochrome P-450 reductase and cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the K$\sb{\rm m}$ of the reductase for cytochrome P-450b or cytochrome P-450c. Modification of carboxyl groups on the reductase with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, and taurine, respectively. The apparent K$\sb{\rm m}$ for cytochrome P-450c or cytochrome P-450b was increased 1.3 to 5.2 fold. There were varied effects on the V$\sb{\rm max}$. There was no significant change in the conformation of the reductase upon chemical modification. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450. Cytochrome P-450 protected 0.8 moles of carboxyl residues on the reductase from being modified with EDC. These protected amino acids on the reductase are presumably involved in binding to cytochrome P-450. The specific peptide containing these amino acids has been identified. ^

The role of menaquinone in the nitrate reductase complex

Membrane bound, respiratory nitrate reductase in Escherichia coli is composed of three subunits, αβγ. The active complex is anchored to the membrane by membrane-integrated γ subunit and can reduce nitrate to nitrite with membrane quinones, (ubiquinone or menaquinone) as physiological electron donors. The transfer of electrons through the complex is thought to involve the sequence: membrane quinols → b-type hemes (γ subunit) → Fe-S centers (β subunit) → molybdopterin (α subunit) → nitrate. The enzyme can be assayed with the artificial electron donor reduced methyl viologen (MVH) which transfers electrons directly to the molybdopterin cofactor. These studies have focused on the possible role of protein-bound menaquinone in the structure and function of this multisubunit complex. ^ Nitrate reductase was purified as two distinct forms; after solubilization of membrane proteins with detergents, purification rendered an αβγ complex (holoenzyme) which catalyzes nitrate reduction with MVH or the quinols analogs, menadiol and duroquinol, as electron donors. Alternatively, heat-treatment of the membranes in the absence of detergents and subsequent purification of the active enzyme produced an αβ complex, which reduces nitrate only with MVH as electron donor. The active αβ dimer was also separated from γ subunit by heat treatment of the holoenzyme. ^ Menaquinone-9 was isolated directly from the purified αβ complex, and identified by mass spectrometry. Based on the composition of the membrane quinone pool, it was concluded that menaquinone-9 is sequestered from the membrane pool in a specifically protein-bound form. ^ The role of the bound menaquinone in the structure-function of nitrate reductase was also investigated, along with its participation in UV-light inactivation of the enzyme. Menaquinone-depleted nitrate reductase from a menaquinone deficient mutant retained activity with all electron donors and it remained sensitive to UV inactivation. However, the MVH-nitrate reductase activity and the rate of UV inactivation of the enzyme were significantly reduced and the optical properties of the enzyme were modified by the absence of the bound menaquinone-9. ^ Menaquinone-9 is not absolutely required for electron transfer in nitrate reductase but it appears to be specifically-bound during assembly of the complex and to enhance the transfer of electrons through the complex. The possible plasticity of the functional electron transfer pathway in nitrate reductase is discussed. ^

Mutation of a single residue in the ba 3 oxidase specifically impairs protonation of the pump site

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^

Function of cardiolipin in mitochondria of Saccharomyces cerevisiae

Cardiolipin and its precursor phosphatidylglycerol, phospholipids found uniquely in membranes engaged in oxidative phosphorylation, play important roles in multimeric complexes of the energy transducing system (ETS) associated with the inner mitochondrial membrane. A combined molecular genetic and biochemical approach was used to more precisely define the role of cardiolipin in cell processes. ^ Strains of yeast Saccharomyces cerevisiae unable to synthesize cardiolipin because of the crd1Δ allele (encodes cardiolipin synthase) with different phenotypes were analyzed to determine which phenotypes are due to lack of cardiolipin. We concluded that many of the severe phenotypes ascribed to cells lacking cardiolipin, particularly when grown at 37°C, are because of the synergistic interaction of the crd1Δ mutation with the reduced expression of the PET56 gene which encodes a component essential for the formation of functional mitochondrial ribosomes. We also demonstrate that much of the reduced mitochondrial function in crd1Δ is because of reduced expression of ETS components at elevated temperature. ^ A crd1Δ mutant of S. cerevisiae has less severe physiological changes than strains lacking both phosphatidylglycerol and cardiolipin due to an increased level of phosphatidylglycerol, which might partially substitute for the cardiolipin-requiring functions. By varying the level of cardiolipin, we were able to correlate phenotypes in a dose-dependent manner with the level of cardiolipin to support more strongly an involvement of cardiolipin in a particular cellular process. There is almost complete lack of a supercomplex composed of cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) in extracts of cardiolipin-lacking mitochondria when compared to wild type cells and the level of supercomplex varies in proportion to the cardiolipin levels. Reduced cardiolipin levels also compromise the growth properties of yeast in a dose-dependent manner suggesting that the loss in growth efficiency is related to a role of cardiolipin that cannot be replaced by phosphatidylglycerol. An independent kinetic approach was performed to compare organization of the respiratory chain in wild-type and cardiolipin-lacking mitochondria. Cardiolipin-lacking mitochondria display kinetic properties for electron transfer between complexes III and IV via cytochrome c consistent with cytochrome c being a freely diffusible carrier, confirming complexes III and IV exist as individual complexes and not associated into a supercomplex in cardiolipin-lacking mitochondria. ^

Temperature tolerance of western Baltic Sea Fucus vesiculosus - growth, photosynthesis and survival

Fucus vesiculosus L. (Phaeophyceae) is the most abundant and hence ecologically most important primary producer, carbon sink and habitat provider in the western Baltic Sea. All F. vesiculosus L. specimens were collected on 23 April 2014 from a depth of 0.2-1 m in the non-tidal Kiel Fjord, western Baltic Sea (54°27'N; 10°12'E), where this species forms dense and almost monospecific stands on stones. After sampling the algal thalli were stored in a refrigerator box with water from the sampling site, transported to Bremerhaven and stored at 10 °C for one day in filtered seawater. Experiments were conducted with vegetative apical tips (6.7±0.5 cm length), the actively growing region of F. vesiculosus, which were randomly selected and cut from 144 different individuals prior to the experiments. These tips were acclimated to laboratory conditions for three days in filtered seawater at 10 °C before the start of the experiment. Furthermore, 30 additional vegetative apices were freeze-dried to document the initial biochemical status of F. vesiculosus in its native habitat. A temperature gradient was installed in a walk-in constant cooling chamber (15 °C) in nine water baths (5, 10, 15, 20, 24, 26, 27, 28 and 29 °C ± 0.1 °C) which were tempered by thermostats (5, 10 and 15 °C: Huber Variostat CC + Pilot ONE, Peter Huber Kältemaschinen GmbH, Offenburg, Germany; 20 and 28 °C: Haake DC3, Thermo Fisher Scientific Inc., Waltham, USA; 24, 26, 27 and 29 °C: Haake DC10). Every temperature treatment consisted of four 2 L glass beakers (n = 4). In each beaker four F. vesiculosus apices were grown in 2 µm-filtered North Sea water diluted with demineralized water in a ratio of 1:1 and enriched with nutrients after Provasoli (1968; 1/10 enrichment), leading to a salinity of about 15.6 which equaled habitat conditions. The algae were exposed to an irradiance of 130 µmol photons m-2 s-1 ±10 % (Powerstar HGI-TS 150 W, OSRAM GmbH, Bad Homburg, Germany) measured at the top of the beaker under a 16:8 h L:D cycle. The media in the beakers was changed every third or fourth day and aerated with artificial air containing 380 ppm CO2 (gas mixing device; HTK Hamburg GmbH, Hamburg, Germany). Before the experiment, the algae were acclimated to the final temperatures in steps of 5 °C for 2 days each, beginning at 10 °C. After 21 days exposure time, three out of four samples per replicate were freeze-dried for further biochemical analyses, and afterwards the thermostats were turned off to reduce the temperature to 16±0.4 °C for another 10 days permitting growth under post-culture conditions.

Anemone abundance and productivity at North Vulcano Island in May 2011

Increased seawater pCO2, and in turn 'ocean acidification' (OA), is predicted to profoundly impact marine ecosystem diversity and function this century. Much research has already focussed on calcifying reef-forming corals (Class: Anthozoa) that appear particularly susceptible to OA via reduced net calcification. However, here we show that OA-like conditions can simultaneously enhance the ecological success of non-calcifying anthozoans, which not only play key ecological and biogeochemical roles in present day benthic ecosystems but also represent a model organism should calcifying anthozoans exist as less calcified (soft-bodied) forms in future oceans. Increased growth (abundance and size) of the sea anemone (Anemonia viridis) population was observed along a natural CO2 gradient at Vulcano, Italy. Both gross photosynthesis (PG) and respiration (R) increased with pCO2 indicating that the increased growth was, at least in part, fuelled by bottom up (CO2 stimulation) of metabolism. The increase of PG outweighed that of R and the genetic identity of the symbiotic microalgae (Symbiodinium spp.) remained unchanged (type A19) suggesting proximity to the vent site relieved CO2 limitation of the anemones' symbiotic microalgal population. Our observations of enhanced productivity with pCO2, which are consistent with previous reports for some calcifying corals, convey an increase in fitness that may enable non-calcifying anthozoans to thrive in future environments, i.e. higher seawater pCO2. Understanding how CO2-enhanced productivity of non- (and less-) calcifying anthozoans applies more widely to tropical ecosystems is a priority where such organisms can dominate benthic ecosystems, in particular following localized anthropogenic stress.

Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer

The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding “molecular excited states.” Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(−)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

ApoNifH functions in iron–molybdenum cofactor synthesis and apodinitrogenase maturation

NifH (dinitrogenase reductase) has three important roles in the nitrogenase enzyme system. In addition to its role as the obligate electron donor to dinitrogenase, NifH is required for the iron–molybdenum cofactor (FeMo-co) synthesis and apodinitrogenase maturation. We have investigated the requirement of the Fe–S cluster of NifH for these processes by preparing apoNifH. The 4Fe–4S cluster of NifH was removed by chelation of the cluster with α, α′-bipyridyl. The resulting apoNifH was tested in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions and was found to function in both these processes. Thus, the presence of a redox active 4Fe–4S cluster in NifH is not required for its function in FeMo-co synthesis and in apodinitrogenase maturation. This, in turn, implies that the role of NifH in these processes is not one of electron transfer or of iron or sulfur donation.

Efficient exchange of the primary quinone acceptor QA in isolated reaction centers of Rhodopseudomonas viridis

A key step in the conversion of solar energy into chemical energy by photosynthetic reaction centers (RCs) occurs at the level of the two quinones, QA and QB, where electron transfer couples to proton transfer. A great deal of our understanding of the mechanisms of these coupled reactions relies on the seminal work of Okamura et al. [Okamura, M. Y., Isaacson, R. A., & Feher, G. (1975) Proc. Natl. Acad. Sci. USA 88, 3491–3495], who were able to extract with detergents the firmly bound ubiquinone QA from the RC of Rhodobacter sphaeroides and reconstitute the site with extraneous quinones. Up to now a comparable protocol was lacking for the RC of Rhodopseudomonas viridis despite the fact that its QA site, which contains 2-methyl-3-nonaprenyl-1,4-naphthoquinone (menaquinone-9), has provided the best x-ray structure available. Fourier transform infrared difference spectroscopy, together with the use of isotopically labeled quinones, can probe the interaction of QA with the RC protein. We establish that a simple incubation procedure of isolated RCs of Rp. viridis with an excess of extraneous quinone allows the menaquinone-9 in the QA site to be almost quantitatively replaced either by vitamin K1, a close analogue of menaquinone-9, or by ubiquinone. To our knowledge, this is the first report of quinone exchange in bacterial photosynthesis. The Fourier transform infrared data on the quinone and semiquinone vibrations show a close similarity in the bonding interactions of vitamin K1 with the protein at the QA site of Rp. viridis and Rb. sphaeroides, whereas for ubiquinone these interactions are significantly different. The results are interpreted in terms of slightly inequivalent quinone–protein interactions by comparison with the crystallographic data available for the QA site of the two RCs.