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BACKGROUND: Both coplanar and noncoplanar polychlorinated biphenyls (PCBs) exhibit neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. Humans genetically have [...]

Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome [cb.sub.562], as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, [D.sub.2]-symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces. doi/10.1073/pnas.0906852107

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 ([P450.sub.BM3]) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides. Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine. biocatalysis | demethylation | evolution | P450 | polysaccharide

The respiratory chain of Eseherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.

Cytochrome [cb.sub.562] is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome [cb.sub.562] refolding triggered using an ultrafast continuous-flow mixer (150 [micro]s dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59--heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome [b.sub.562] folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps. four-helix bundle | minimal frustration | protein folding | time-resolved fluorescence energy transfer | tryptophan

We hypothesize that [A.sub.2A] adenosine receptors ([A.sub.2A] AR) promote aortic relaxation in mice through cytochrome P450 (CYP)-epoxygenases and help to avoid salt sensitivity. Aortas from male mice maintained on a high-salt (HS; 7% NaCl) or normal-salt (NS; 0.45% NaCL) diet for 4-5 wks were used. Concentration-response curves ([10.sup.-11]-[10.sup.-5] M) for 5'-N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine analog) and CGS 21680 ([A.sub.2A] AR agonist) were obtained with different antagonists including ZM 241385 ([A.sub.2A] AR antagonist; [10.sup.-6] M), SCH 58261 ([A.sub.2A] AR antagonist; [10.sup.-6] M), [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME; endothelial nitric oxide synthase inhibitor; [10.sup.-4] M) and inhibitors including methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; CYP epoxygenases inhibitor; [10.sup.-5]M), 14,15-epoxyeicosa-5(z)-enoic acid (14,15-EEZE; EET antagonist; [10.sup.-5]M), dibromo-dodecenyl-methylsulfimide (DDMS; CYP4A inhibitor; [10.sup.-5]M), and HET0016 (20-HETE inhibitor; [10.sup.-5]M). At [10.sup.-7] M of NECA, significant relaxation in HS (+22.58 [+ or -] 3.12%) was observed compared with contraction in NS (-10.62 [+ or -] 6.27%, P < 0.05). ZM 241385 changed the NECA response to contraction (P < 0.05) in HS. At [10.sup.-7] M of CGS 21680, significant relaxation in HS (+32.04 [+ or -] 3.08%) was observed compared with NS (+ 10.45 [+ or -] 1.34%, P < 0.05). SCH 58261, L-NAME, MS-PPOH, and 14,15-EEZE changed the CGS 21680-induced relaxation to contraction (P < 0.05) in HS. Interestingly, DDMS and HET0016 changed CGS 21680 response to relaxation (P < 0.05) in NS; however, there was no significant difference found between DDMS, HET0016-treated HS and NS vs. nontreated HS group (P > 0.05). CYP2C29 protein was 55% and 74% upregulated in HS vs. NS (P < 0.05) mice aorta and kidney, respectively. CYP4A protein was 30.30% and 35.70% upregulated in NS vs. HS (P < 0.05) mice aorta and kidneys, respectively. [A.sub.1] AR was downregulated, whereas [A.sub.2A] AR was upregulated in HS compared with NS. These data suggest that HS may activate CYP2C29 via [A.sub.2A] AR, causing relaxation, whereas NS may contribute to the upregulation of CYP4A causing contraction. vasodilation; vasoconstriction

MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-[PK.sub.1] cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-[PK.sub.1] cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release (P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 [micro]M) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 [micro]M) also inhibited cytotoxicity significantly (P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase (P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells (P < 0.05). This was abolished in the presence of HET-0016 (P < 0.05) or MnTMPyP (P < 0.01). These results demonstrate that 20-HETE over-expression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals. apoptosis; cytochrome P-450 4A10; cytochrome P-450 4A12; LLC-[PK.sub.1]; lentiviral vectors; kidney; HET-0016; superoxide; [0.sub.2]

Kruppel-like factors (KLFs) are important Sp1-like eukaryotic transcriptional proteins. The LDLR, STAR, and CYP11A genes exhibit GC-rich Sp1-like sites, which have the potential to bind KLFs in multiprotein complexes. We now report that KLF4, KLF9, and KLF13 transcripts are expressed in and regulate ovarian cells. KLF4 and 13, but not KLF9, mRNA expression was induced and then repressed over time (P < 0.001). Combined LH and IGF-I stimulation increased KLF4 mRNA at 2 h (P < 0.01), whereas LH decreased KLF13 mRNA at 6 h (P < 0.05), and IGF-I reduced KLF13 at 24 h (P < 0.01) compared with untreated control. KLF9 was not regulated by either hormone. Transient transfection of KLF4, KLF9, and KLF13 suppressed LDLR/luc, StAR/luc, and CYP11A/luc by 80-90% (P < 0.001). Histone-deacetylase (HDAC) inhibitors stimulated LDLR/luc five- to sixfold and StAR/luc and CYP11A/luc activity twofold (P < 0.001) and partially reversed suppression by all three KLFs (P < 0.001). Deletion of the zinc finger domain of KLF13 abrogated repression of LDLR/ luc. Lentiviral overexpression of the KLF13 gene suppressed LDLR mRNA (P < 0.001) and CYP11A mRNA (P = 0.003) but increased StAR mRNA (P = 0.007). Collectively, these data suggest that KLFs may recruit inhibitory complexes containing HDAC corepressors, thereby repressing LDLR and CYP11A transcription. Conversely, KLF13 may recruit unknown coactivators or stabilize StAR mRNA, thereby explaining enhancement of in situ StAR gene expression. These data introduce new potent gonadal transregulators of genes encoding proteins that mediate sterol uptake and steroid biosynthesis. sterol; gonad; luteal; low-density lipoprotein receptor; steroidogenic acute regulatory protein; cytochrome P-450 cholesterol side-chain cleavage

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 rain global ischemia or following 40 min of perfusion alone as controls. [H.sub.2][O.sub.2] release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of [H.sub.2][O.sub.2] was increased by 178 [+ or -] 14% and 179 [+ or -] 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, [H.sub.2][O.sub.2] increased by 272 [+ or-] 22% and 171 [+ or -] 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased [H.sub.2][O.sub.2] production by 179 [+ or -] 24% and 155 [+ or -] 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of [H.sub.2][O.sub.2] from complex IH. Thus ischemic damage to the ETC increased both the capacity and the net production of [H.sub.2][O.sub.2] from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury. nicotinamide adenine dinucleotide:ubiquinone oxidoreductase (complex I), ubiquinol:cytochrome c oxidoreductase (complex III), cytochrome c; ischemia

Biological electron transfer (eT) between redox-active cofactors is thought to occur by quantum-mechanical tunneling. However, in many cases the observed rate is limited by other reactions coupled to eT, such as proton transfer, conformational changes, or catalytic chemistry at an active site. A prominent example of this phenomenon is the eT between the heme groups of mitochondrial cytochrome c oxidase, which has been reported to take place in several different time domains. The question of whether pure eT tunneling in the nanosecond regime between the heme groups can be observed has been the subject of some experimental controversy. Here, we report direct observations of eT between the heme groups of the quinol oxidase cytochrome [bo.sub.3] from Escherichia coil, where the reaction is initiated by photolysis of carbon monoxide from heme [o.sub.3]. eT from CO-dissociated ferrous heme [o.sub.3] to the low-spin ferric heme b takes place at a rate of [(1.2 ns)-1] at 20[degrees]C as determined by optical spectroscopy. These results establish hemeheme electron tunneling in the [bo.sub.3] enzyme, a bacterial relative to the mitochondrial cytochrome c oxidase. The properties of eT between the closely lying heme groups in the heme-copper oxidases are discussed in terms of the reorganization energy for the process, and two methods for assessing the rate of electron tunneling are presented. biological electron transfer | heme-copper oxidases | Marcus theory | Moser-Dutton ruler | ultrafast spectroscopy

Haem [d.sub.1] is a unique tetrapyrrole molecule that serves as a prosthetic group of cytochrome [cd.sub.1], which reduces nitrite to nitric oxide during the process of denitrification. Very little information is available regarding the biosynthesis of haem [d.sub.1]. The extreme difficulty in studying the haem [d.sub.1] biosynthetic pathway can be partly attributed to the lack of a theoretical basis for experimental investigation. We report here a gene cluster encoding enzymes involved in the biosynthesis of haem [d.sub.1] in two heliobacterial species, Heliobacillus mobilis and Heliophilum fasciatum. The gene organization of the cluster is conserved between the two species, and contains a complete set of genes that lead to the biosynthesis of uroporphyrinogen III and genes thought to be involved in the late steps of haem [d.sub.1] biosynthesis. Detailed bioinformatics analysis of some of the proteins encoded in the gene cluster revealed important clues to the precise biochemical roles of the proteins in the biosynthesis of haem [d.sub.1], as well as the membrane transport and insertion of haem [d.sub.1] into an apocytochrome during the maturation of cytochrome [cd.sub.1].

Addition of Pco (~350 Torr) to a normoxic medium ([Po.sub.2] of ~130 Torr) was used to investigate the relationship between carotid body (CB) sensory discharge and expression of hypoxia-inducible factor 1[alpha] (HIF-1[alpha]) in glomus cells. Afferent electrical activity measured for in vitro-perfused rat CB increased rapidly (1-2 s) with addition of high CO (Pco of ~350 Torr; [Po.sub.2] of ~130 Torr), and this increase was fully reversed by white light. At submaximal light intensities, the extent of reversal was much greater for monochromatic light at 430 and 590 nm than for light at 450, 550, and 610 nm. This wavelength dependence is consistent with the action spectrum of the CO compound of mitochondrial cytochrome [a.sub.3]. Interestingly, when isolated glomus cells cultured for 45 min in the presence of high CO (Pco of ~350 Torr; [Po.sub.2] of ~130 Torr) in the dark, the levels of HIF-1[alpha], which turn over slowly (many minutes), increased. This increase was not observed if the cells were illuminated with white light during the incubation. Monochromatic light at 430+ and 590-nm light was much more effective than that at 450, 550, and 610 nm in blocking the CO-induced increase in HIF-1[alpha], as was the case for chemoreceptor discharge. Although the changes in HIF-1[alpha] take minutes and those for CB neural activity occur in 1-2 s, the similar responses to CO and light suggest that the oxygen sensor is the same (mitochondrial cytochrome [a.sub.3]). carbon monoxide; carotid body; cytochrome [a.sub.3]; hypoxia-inducible factor 1[alpha]; mitochondria; sensory discharge

Details are presented of the structural analysis of the cytochrome [b.sub.6]f complex from the thermophilic cyanobacterium, Mastigocladus laminosus, in the presence of the electrochemically positive (p)-side quinone analogue inhibitor, 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). One DBMIB binding site was found. This site is peripheral to the quinone binding space defined by the binding sites of other p-side inhibitors previously resolved in cytochrome [bc.sub.1]/[b.sub.6]f complexes. This high-affinity site resides in a p-side interfacial niche bounded by cytochrome f, subunit IV, and cytochrome [b.sub.6], is close (8 [Angstrom]) to the p-side heme b, but distant (19 A) from the [2Fe-2S] cluster. No significant electron density associated with the DBMIB was found elsewhere in the structure. However, the site at which DBMIB can inhibit light-induced redox turnover is within a few A of the [2Fe-2S] cluster, as shown by the absence of inhibition in mutants of Synechococcus sp. PCC 7002 at iron sulfur protein-Leu-111 near the cluster. The ability of a minimum amount of initially oxidized DBMIB to inhibit turnover of WT complex after a second light flash implies that there is a light-activated movement of DBMIB from the distal peripheral site to an inhibitory site proximal to the [2Fe-2S] cluster. Together with the necessary passage of quinone/quinol through the small [Q.sub.p] portal in the complex, it is seen that transmembrane traffic of quinone-like molecules through the core of cytochrome be complexes can be labyrinthine. photosynthetic membrane proteins | energy transduction | transmembrane traffic

Protein folding is uniquely characterized as a dynamic process by the tremendous conformational heterogeneity of the unfolded state. The kinetics of the fast folding process imply that it proceeds from a conformational ensemble that is not in equilibrium with the bulk of protein conformers during the time required to completely reduce the sample ~100Mus.

The measurement of the dissociation constants for the binding of Rhodobacter capsulatus cytochrome [c.sub.2] and its K93P mutant to the cytochrome b[c.sub.1] complex embedded in a phospholipid bilyaer reveal that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome [c.sub.2] to cytochrome [c.sub.1].

The construction and the kinetic and crystallographic characterization of the A251C:S430C double mutant form of flavocytochrome C(sub 3) is described. The results show that clamp domain mobility is affected by catalysis but is not critical for the regulation of substrate access/product removal and the altered kinetic characteristics in the oxidized mutant enzyme are due to the perturbation of active site rearrangement during catalysis as a consequence of disulfide bond formation.

N-Acetylmicroperoxidase-8 contains heme and residues 14-21 of horse mitochondrial cytochrome c(cyt c). The two thioether bonds linking protein to heme in cyt c are present in N-Acetylmicroperoxidase-8, and the native axial ligand His-18 remains coordinated to iron.

P450 1A2 enzymes are involved in the oxidation of many drugs, pollutants, and carcinogens and many endogenous compounds. The P450's have limited selectivity in physiological processes but the P450's oxidizing xenobiotics ability and its catalytic selectivity is of paramount importance in the development of new drugs.