1. Heteroactivator effects on the coupling and spin state equilibrium of CYP2C9
Charles W. Locusona, Peter M. Gannettb, Timothy S. Tracya, ,
The cytochromes P450 are capable of oxidizing a variety of xenobiotics. Binding of a small molecule heteroactivator to a P450 can alter the coupling of substrate oxidation during P450 catalysis, but the degree to which coupling or shunting via one of the three catalytic cycle branch points is linked to the heteroactivator-modified position of bound substrate is unknown. Using reconstituted CYP2C9, stoichiometric measurements were gathered with three substrates and two classes of heteroactivators to further understand the mechanisms involved in heteroactivation. Heteroactivation of P450 metabolism appeared to involve, but not require, changes in coupling and that increased uncoupling to a specific byproduct like H2O2 does not necessarily correlate to the degree of coupling. In addition, spectroscopy demonstrated that every heteroactivator tested influenced the spin equilibrium of the heme iron even in the presence of saturating substrate suggesting that both substrate proximity and the ability to desolvate the heme can be involved in heteroactivation.
2. Functional Analysis of Phenylalanine Residues in the Active Site of Cytochrome P450 2C9
Carrie M. Mosher ‡, Matthew A. Hummel §, Timothy S. Tracy § and Allan E. Rettie
The two published crystal structures of cytochrome P450 2C9, complexed with (S)-warfarin or flurbiprofen, implicate a cluster of three active site phenylalanine residues (F100, F114, F476) in ligand binding. However, these three residues appear to interact differently with these two ligands based on the static crystal structures. To elucidate the importance of CYP2C9’s active site phenylalanines on substrate binding, orientation, and catalytic turnover, a series of leucine and tryptophan mutants were constructed and their interactions with (S)-warfarin and (S)-flurbiprofen examined. The F100→L mutation had minor effects on substrate binding and metabolism of each substrate. In contrast, the F114L and F476L mutants exhibited substantially reduced (S)-warfarin metabolism and altered hydroxy metabolite profiles but only modestly decreased nonsteroidal antiinflammatory drug (NSAID) turnover while maintaining product regioselectivity. The F114→W and F476→W mutations also had opposing effects on (S)-warfarin versus NSAID turnover. Notably, the F476W mutant increased the efficiency of (S)-warfarin metabolism 5-fold, yet decreased the efficiency of (S)-flurbiprofen turnover 20-fold. 1H NMR T1 relaxation studies suggested a slightly closer positioning of (S)-warfarin to the heme in the F476W mutant relative to the wild-type enzyme, and stoichiometry studies indicated enhanced coupling of reducing equivalents to product formation for (S)-warfarin, again in contrast to effects observed with (S)-flurbiprofen. These data demonstrate that F114 and F476, but not F100, influence (S)-warfarin’s catalytic orientation. Differential interactions of F476 mutants with the two substrates suggest that their catalytically productive binding modes are not superimposable.
3. Rates and Extent of Reduction of Fe(III) Compounds and O2 by Humic Substances
Iris Bauer and Andreas Kappler
Humic substances (HS) can be reduced by microorganisms and oxidized by electron acceptors such as Fe(III) or O2. However, redox reactions between HS and highly crystalline Fe(III) minerals and O2 have not yet been quantified. We therefore determined the rates and extent of goethite and hematite reduction by HS in comparison to those of dissolved and poorly crystalline Fe(III) compounds and O2. Although nonreduced HS transferred significant amounts of electrons only to dissolved Fe(III) citrate and ferrihydrite, reduced HS additionally reduced goethite and hematite. The extent of reduction depended on the redox potentials of the Fe(III) compounds. Fewer electrons were transferred from HS to O2 than to Fe(III) despite the more positive redox potential of the O2/H2O redox couple. Reoxidation of reduced HS by O2 took place within minutes and yielded reoxidized HS that were still more reduced than nonreduced HS, indicating that some reduced moieties in HS are protected from reoxidation by O2. Our data suggests (i) reduction of crystalline Fe(III) minerals by reduced HS has to be considered in the environmental electron transfer network, (ii) exposure of reduced HS to O2 does not reoxidize HS completely within short time frames, and therefore, (iii) HS electron shuttling to Fe(III) can occur even in the presence of O2.
4. Circadian rhythms in metabolic variables in Caenorhabditis elegans
María Laura Migliori, Sergio H. Simonetta, Andrés Romanowski, Diego A. Golombek
Circadian rhythms govern a wide variety of physiological and metabolic functions in most organisms through neural networks, hormones and gene expression.
In this work, we studied the circadian variation in metabolic variables of adult C. elegans such as food consumption, pharyngeal contractions, defecation and oxygen consumption. Feeding behavior was clearly rhythmic under LD conditions, with a non-significant trend under DD conditions. In addition, a daily and circadian variation in muscle contraction of the pharynx was observed. Oxygen consumption also showed a circadian fluctuation with a maximum in the middle of the night (a peak was found around ZT18/CT18). Furthermore, defecation behavior also showed a daily variation in the N2 strain (wild type). This work demonstrates that in the adult nematode C. elegans metabolic variables vary daily. In summary, our results will allow us to take full advantage of this widely used animal model (including research in genetics, ageing and developmental biology) for studies in Chronobiology.
5. Mutagenesis of tyrosine residues within helix VII in subunit I of the cytochrome cbb3 oxidase from Rhodobacter capsulatus
Mehmet Öztürk and Nicholas J. Watmough
The cbb 3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. The cbb 3 oxidases are missing an active-site tyrosine residue that is absolutely conserved in all A and B-type heme-copper oxidases. This tyrosine is known to play a critical role in the catalytic mechanisms of A and B-type oxidases. The absence of this tyrosine in the cbb 3 oxidases raises the possibility that the cbb 3 oxidases utilize a different catalytic mechanism from that of the other members of the superfamily, or have this conserved residue in different helices. Recently sequence comparisons indicate that, a tyrosine residues that might be analogous to the active-site tyrosine in other oxidases are present in the cbb 3 oxidases but these tyrosines originates from a different transmembrane helix within the protein. In this research, three conserved tyrosine residues, Y294, Y308 and Y318, in helix VII were substituted for phenylalanine. Y318F mutant in the Rhodobacter capsulatus oxidase resulted in a fully assembled enzyme with nativelike structure and activity, but Y294F mutant is not assembled and have a catalytic activity. On the other hand, Y308F mutant is fully assembled enzyme with nativelike structure, but lacking catalytic activity. This result indicates that Y308 should be crucial in catalytic activity of the cbb 3 oxidase of R. capsulatus. These findings support the assumption that all of the heme-copper oxidases utilize the same catalytic mechanism and provide a residue originates from different places within the primary sequence for different members of the same superfamily.
6. The zebrafish embryo as a dynamic model of anoxia tolerance
Bryce A. Mendelsohn, Bethany L. Kassebaum, Jonathan D. Gitlin
Developing organisms depend upon a delicate balance in the supply and demand of energy to adapt to variable oxygen availability, although the essential mechanisms determining such adaptation remain elusive. In this study, we examine reversible anoxic arrest and dynamic bioenergetic transitions during zebrafish development. Our data reveal that the duration of anoxic viability corresponds to the developmental stage and anaerobic metabolic rate. Diverse chemical inhibitors of mitochondrial oxidative phosphorylation induce a similar arrest in normoxic embryos, suggesting a pathway responsive to perturbations in aerobic energy production rather than molecular oxygen. Consistent with this concept, arrest is accompanied by rapid activation of the energy-sensing AMP-activated protein kinase pathway, demonstrating a potential link between the sensing of energy status and adaptation to oxygen availability. These observations permit mechanistic insight into energy homeostasis during development that now enable genetic and small molecule screens in this vertebrate model of anoxia tolerance. Developmental Dynamics 237:1780–1788, 2008. © 2008 Wiley-Liss, Inc.
7. Coordination of development and metabolism in the pre-midblastula transition zebrafish embryo
Bryce A. Mendelsohn, Jonathan D. Gitlin
To define the mechanisms that coordinate early embryonic development and metabolism, we have examined the response of zebrafish embryos to anoxia before the midblastula transition. Our findings reveal that anoxic pre-midblastula transition embryos slow the cell cycle, arrest before the midblastula transition and can recover normally if restored to a normoxic environment. Analyses of respiratory rates reveal that pre-midblastula transition embryos are less reliant on oxidative phosphorylation than older embryos. Interestingly, arrest in anoxia occurs despite inhibition of zygotic transcription, revealing a central role for maternal factors in the response to energy limitation. Consistent with this concept, we demonstrate that the posttranslational energy-sensing AMP-activated protein kinase pathway is activated in anoxia in pre-midblastula transition embryos. Taken together, these findings demonstrate a maternal program capable of coordinating developmental rate and metabolism in the absence of transcription-based pathways or cell cycle checkpoints. Developmental Dynamics 237:1789–1798, 2008. © 2008 Wiley-Liss, Inc.
8. Generation and characterization of His-tagged-PsbA-expressing transformants of Chlamydomonas reinhardtii that are capable of photoautotrophic growth
Janewit Wongratana, Thanate Juntadech, Chutima Sereeruk, Chanan Angsuthanasombat and Kittisak Yokthongwattana
Histidine tags attached to subunits of photosystem II (PSII) have proven to be very useful tools for isolation and purification of the complex for investigation of its components. However, it has been reported that Chlamydomonas reinhardtii transformants carrying N-terminal histidine-tagged version of the PSII D1 reaction center protein could not grow photoautotrophically. We report here a successful generation of C. reinhardtii transformants expressing histidine-tagged version of the PsbA protein that are capable of photoautotrophic growth. Biochemical and physiological analyses revealed that the histidine tag present at the N terminus of the D1 subunit did not cause total instability to the PSII complex as assessed by their phototrophic growth and oxygen evolution capability. Simple one-step affinity column chromatography also revealed that the histidine-tagged D1 subunit as well as its associated proteins could be effectively purified. These transformants could potentially serve as very good tools for the study of the PSII complex particularly the D1 protein.
9. Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria
Joonho Park, Nicholas Khuu, Alexander S. M. Howard, Robert T. Mullen, William C. Plaxton
Keywords: phosphoenolpyruvate carboxylase [PEPC (Ppc)];Ricinus communis (castor oil seeds);fluorescent protein imaging;protein–protein interactions;mitochondria;oil seed metabolism
Phosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC’s unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO2 while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.
10. Survival of Campylobacter jejuni in co-culture with Acanthamoeba castellanii: role of amoeba-mediated depletion of dissolved oxygen
Xuan Thanh Bui1, Anne Winding, Klaus Qvortrup, Anders Wolff, Dang Duong Bang, Carole Creuzenet
SummaryCampylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C. jejuni in A. castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A. castellanii promoted the extracellular growth of C. jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae – bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C. jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C. jejuni did not promote the growth of C. jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae – bacteria contact were much lower than those observed with culture media or with C. jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C. jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A. castellanii as the major contributor for the observed amoeba-mediated growth enhancement.