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Background: The prognosis for most patients with supraventricular tachycardia (SVT) is thought to be excellent. However, the fatality rate in children with SVT is poorly defined and there are no large-scale, multicenter studies to support this commonly held belief., Methods: We reviewed an administrative database containing inpatient records from 41 stand-alone children's hospitals and identified all hospitalized patients younger than 25 years with a discharge diagnosis of SVT between January 2003 and September 2008., Results: We analyzed the index hospitalization of 1,755 patients with the discharge diagnosis of SVT, of whom 58% were male and 52% had comorbid structural heart disease (SHD). There were a total of 68 patient deaths (4%) with 56 of 68 (82%) of the deaths occurring in patients with SHD. In total, 6% of the patients with SHD died, whereas only 1% of the patients without SHD died. Regression analysis revealed that case fatality is increased in patients less than 1 month of age (OR 2.41, 95% CI 1.35 to 4.32), in patients with SHD (OR 2.67, 95% CI 1.22 to 5.80), and in those having cardiomyopathy with (OR 6.72, 95% CI 1.79 to 25.28) and without SHD (OR 6.74, 95% CI 1.67 to 27.26)., Conclusions: The case fatality in patients who have SVT without SHD is low. It is increased in the very young and in those with comorbid cardiovascular disease. Case fatality is highest in patients with a cardiomyopathy regardless of SHD., (©2011, The Authors. Journal compilation ©2011 Wiley Periodicals, Inc.)

In many energy transducing systems which couple electron and proton transport, for example, bacterial photosynthetic reaction center, cytochrome bc(1)-complex (complex III) and E. coli quinol oxidase (cytochrome bo(3) complex), two protein-associated quinone molecules are known to work together. T. Ohnishi and her collaborators reported that two distinct semiquinone species also play important roles in NADH-ubiquinone oxidoreductase (complex I). They were called SQ(Nf) (fast relaxing semiquinone) and SQ(Ns) (slow relaxing semiquinone). It was proposed that Q(Nf) serves as a "direct" proton carrier in the semiquinone-gated proton pump (Ohnishi and Salerno, FEBS Letters 579 (2005) 4555), while Q(Ns) works as a converter between one-electron and two-electron transport processes. This communication presents a revised hypothesis in which Q(Nf) plays a role in a "direct" redox-driven proton pump, while Q(Ns) triggers an "indirect" conformation-driven proton pump. Q(Nf) and Q(Ns) together serve as (1e(-)/2e(-)) converter, for the transfer of reducing equivalent to the Q-pool., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Rationale: GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH(4)), an essential cofactor for NO synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH(4) via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH(4) levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH(4) production in response to laminar shear., Objective: We sought to define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP., Methods and Results: Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH(4) levels, and NO production in human endothelial cells. Laminar, but not oscillatory shear stress, caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH(4) levels in vivo., Conclusions: These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH(4) to be produced in response to laminar shear stress.

The temporal aspects of signaling are critical to the function of signals in communications, feedback regulation and control. The production and transduction of biological signals by enzymes comprises an area of central importance and rapid progress in the biomedical sciences. Treatment of signaling enzymes almost universally employs steady-state analyses that are suitable for mass catalysis but inappropriate for components in an information channel or a feedback/control system. In the present study, we show that, at 37 degrees C, neuronal nitric oxide synthase (EC 1.14.13.39) is progressively inhibited by the formation of an inhibited state during the first few turnovers (approximately 200 ms) after the initiation of catalysis, leading to pulse formation of nitric oxide. The general mechanism may be of wide importance in biological signaling.

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -L-[(14)C]arginine-to-L-[(14)C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN(2)-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN(2)-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN(2)-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN(2)-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with L-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the "base-off" conformation of the lower axial dimethylbenzimidazole ligand. CN(2)-Cbi inhibited interferon-gamma-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.

Supraventricular tachycardia is the most common rhythm disturbance in children. We reviewed the spectrum of this common rhythm disorder from symptom recognition and epidemiology to management, with special attention to advancements in the available treatment options.

Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K(m) for H(2)O(2) of 1.5 mM. When co-expressed in cells, VPO1 can use H(2)O(2) produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system.

Left ventricular noncompaction (LVNC) is an uncommon disorder that has recently been recognized as a distinct cardiomyopathy. LVNC is thought to result from an arrest in the normal process of myocardial compaction. The association of Wolff-Parkinson-White with noncompaction of the left ventricle is well recognized. Sinus bradycardia has also been associated with LVNC, although less frequently than that of Wolff-Parkinson-White. We report an infant with LVNC, Wolff-Parkinson-White, and progressive sinus bradycardia who had a myocardial vascular abnormality in the region of the sinus node evident on autopsy. We propose that the progressive nature of the conduction system abnormality was as a result of abnormal angiogenesis.

The established paradigm in understanding and describing enzyme activity uses formalisms based on steady-state assumptions, including Michaelis-Menten and King-Altman approaches. These are appropriate for enzymes operating under steady-state conditions. Signal generating enzymes transfer information, rather than material. Because the information capacity of a signal channel depends on frequency, steady-state descriptions may not be appropriate. Recently, Stuehr and coworkers described a novel product inhibition mechanism for NO synthases. Simulations presented here suggest that at physiological temperatures neuronal nitric oxide synthase produces sharp pulses of NO, consistent with its signaling function. These temporal pulses greatly restrict the effective spatial range of NO signaling.

Purpose: The sequentially variable COOH-terminal region of small heat shock protein superfamily members usually contains a conserved IXI/V feature where X is typically a proline. When present in solved sHsp crystal structures (e.g. MjHsp16.5 and wheat Hsp16.9), this short sequence forms an isolated beta strand apparently involved in the alignment of dimers into larger oligomers. Because it is a common feature of many sHsp family members, it is possible that this triad has a similar role in alphaA-crystallin. This study was undertaken to determine the contribution of this conserved triad to the quaternary structure and function of alphaA-crystallin., Methods: A series of site-directed mutants was generated in both wild type alphaA and in an alphaA deletion mutant lacking the NH2-terminal residues 1-50. After overexpression and purification, each protein's oligomer size was characterized by size-exclusion fast protein liquid chromatography (FPLC), thermal transition temperature by non-denaturing composite gel electrophoresis, and chaperone activity by the inhibition of DL-dithiothreitol (DTT)-induced insulin aggregation., Results: Using the alphaA-crystallin NH2-deletion mutant, the hydrophobic triad was changed from IPV to TPT, GPG, IGV, ITV, or GGG. All six D51 mutants associated into tetramers with small amounts of dimer and monomer also present. Chaperone-like activity was reduced but not eliminated in some of these triad mutants with GGG and ITV the most strongly affected. Similar modifications to wild type alphaA-crystallin (IPV to ITV, IGV, or GGG) restored oligomer sizes similar, but not identical to, native alphaA-crystallin, with additional small amounts of tetramer and dimer. Interestingly, equivalent mutants of wild type alphaA-crystallin did not have reduced chaperone-like activity but differed considerably in their thermal transition temperatures., Conclusions: The conserved COOH-terminal triad does not appear to have a strong effect on the steady-state aggregation of wild type alphaA-crystallin or its 50-residue deletion mutant at 25 degrees C. However, it can exert a considerable effect on chaperone-like activity in the absence of the NH2-terminal 50-residue sequence extension and can influence the thermal transition temperature in its presence. These results suggest that the conserved triad in alphaA-crystallin contributes to the stability of higher order oligomers but is not essential for the formation of tetramers.

Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKIIdelta(2) with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with gamma-tubulin. Furthermore, CaMKIIdelta(2) coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca(2+)-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKIIdelta(2) mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKIIdelta(2) using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKIIdelta(2) may be an important regulator of iNOS trafficking and activity in VSMCs.

Intraprotein interdomain electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Our previous laser flash photolysis studies have provided a direct determination of the kinetics of IET between the FMN and heme domains in truncated oxyFMN constructs of rat neuronal NOS (nNOS) and murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present [Feng, C. J.; Tollin, G.; Holliday, M. A.; Thomas, C.; Salerno, J. C.; Enemark, J. H.; Ghosh, D. K. Biochemistry 2006, 45, 6354-6362. Feng, C. J.; Thomas, C.; Holliday, M. A.; Tollin, G.; Salerno, J. C.; Ghosh, D. K.; Enemark, J. H. J. Am. Chem. Soc. 2006, 128, 3808-3811]. Here, we report the kinetics of IET between the FMN and heme domains in a rat nNOS holoenzyme in the presence and absence of added CaM using laser flash photolysis of CO dissociation in comparative studies on partially reduced NOS and a single domain NOS oxygenase construct. The IET rate constant in the presence of CaM is 36 s-1, whereas no IET was observed in the absence of CaM. The kinetics reported here are about an order of magnitude slower than the kinetics in a rat nNOS oxyFMN construct with added CaM (262 s-1). We attribute the slower IET between FMN and heme in the holoenzyme to the additional step of dissociation of the FMN domain from the reductase complex before reassociation with the oxygenase domain to form the electron-transfer competent output state complex. This work provides the first direct measurement of CaM-controlled electron transfer between catalytically significant redox couples of FMN and heme in a nNOS holoenzyme.

The crystal structure of the neuronal nitric-oxide synthase (nNOS) NADPH/FAD binding domain indicated that Ser-1176 is within hydrogen bonding distance of Asp-1393 and the O4 atom of FAD and is also near the N5 atom of FAD (3.7 A). This serine residue is conserved in most of the ferredoxin-NADP+ reductase family of proteins and is important in electron transfer. In the present study, the homologous serines of both nNOS (Ser-1176) and endothelial nitric-oxide synthase (eNOS) (Ser-942) were mutated to threonine and alanine. Both substitutions yielded proteins that exhibited decreased rates of electron transfer through the flavin domains, in the presence and absence of Ca2+/CaM, as measured by reduction of potassium ferricyanide and cytochrome c. Rapid kinetics measurements of flavin reduction of all the mutants also showed a decrease in the rate of flavin reduction, in the absence and presence of Ca2+/CaM, as compared with the wild type proteins. The serine to alanine substitution caused both nNOS and eNOS to synthesize NO more slowly; however, the threonine mutants gave equal or slightly higher rates of NO production compared with the wild type enzymes. The midpoint redox potential measurements of all the redox centers revealed that wild type and threonine mutants of both nNOS and eNOS are very similar. However, the redox potentials of the FMN/FMNH* couple for alanine substitutions of both nNOS and eNOS are >100 mV higher than those of wild type proteins and are positive. These data presented here suggest that hydrogen bonding of the hydroxyl group of serine or threonine with the isoalloxazine ring of FAD and with the amino acids in its immediate milieu, particularly nNOS Asp-1393, affects the redox potentials of various flavin states, influencing the rate of electron transfer.

Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s(-)(1)) was increased approximately 10-fold compared to that in the absence of CaM (22 s(-)(1)). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH. radical signals in inducible nitric-oxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquinone couple = +70 mV, FMN semiquinone/hydroquinone couple = -180 mV, and heme = -180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.

We report here that NADPH analogs such as 2'5'ADP, ATP, and 2'AMP paradoxically activate constitutive calcium/calmodulin regulated nitric oxide synthases (cNOS), including the endothelial isoform (eNOS) and the neuronal isoform (nNOS). These activators compete with NADPH by filling the binding site of the adenine moiety of NADPH, but do not occupy the entire NADPH binding domain. Effects of these analogs on cNOS's include increasing the electron transfer rate to external acceptors, as assessed by cytochrome c reductase activity in the absence of calmodulin. In addition, NO synthase activity in the presence of calmodulin (with or without added calcium) was increased by the addition of NADPH analogs. In contrast, the same NADPH analogs inhibit iNOS, the calcium insensitive inducible isoform, which lacks control elements found in constitutive isoforms. Because ATP and ADP are among the effective activators of cNOS isoforms, these effects may be physiologically relevant.

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.

Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.

A novel mechanism for proton/electron transfer is proposed for NADH-quinone oxidoreductase (complex I) based on the following findings: (1) EPR signals of the protein-bound fast-relaxing semiquinone anion radicals (abbreviated as Q(Nf)-) are observable only in the presence of proton-transmembrane electrochemical potential; (2) Iron-sulfur cluster N2 and Q(Nf)- are directly spin-coupled; and (3) The projection of the interspin vector extends only 5A along the membrane normal [Yano, T., Dunham, W.R. and Ohnishi, T. (2005) Biochemistry, 44, 1744-1754]. We propose that the proton pump is operated by redox-driven conformational changes of the quinone binding protein. In the input state, semiquinone is reduced to quinol, acquiring two protons from the N (matrix) side of the mitochondrial inner membrane and an electron from the low potential (NADH) side of the respiratory chain. A conformational change brings the protons into position for release at the P (inter-membrane space) side of the membrane via a proton-well. Concomitantly, an electron is donated to the quinone pool at the high potential side of the coupling site. The system then returns to the original state to repeat the cycle. This hypothesis provides a useful frame work for further investigation of the mechanism of proton translocation in complex I.

Purpose: To assess the role of the NH2-terminal in alphaA-crystallin folding and chaperone-like activity., Methods: Two NH2-terminal deletion mutants of alphaA-crystallin were generated by standard mutagenesis methods, one with and one without a leader sequence in place of the first 50 residues. Aggregate size of each before and after thermal stress was assessed by FPLC, and chaperone-like activity was assessed using DTT-induced insulin denaturation., Results: Both mutants assemble primarily into tetramers, and both exhibit similar levels of chaperone-like activity, but are less protective than recombinant alphaA-crystallin. After a cycle of heat stress to 70 degrees C, tetramers of the mutant without the leader sequence dissociate into dimers and monomers and show severely reduced chaperone-like activity. In contrast, the mutant with the leader sequence retains its tetrameric form and its chaperone-like activity., Conclusions: The NH2-terminal region is an important determinant of alpha-crystallin aggregate size, but is not required for folding of the alpha-crystallin domain, since the aggregate size and chaperone-like activity of the two mutants at room temperature are essentially the same. The leader sequence appears to increase the thermal stability of the alpha-crystallin domain and/or to contribute to the reformation of the active form after cooling, suggesting that the native NH2-terminal also plays a role in alpha-crystallin's resistance to environmental stress.

The small heat shock protein superfamily is composed of proteins from throughout the phylogenetic spectrum that are induced upon environmental stress. Their structural stability under stress derives in large part from the central region of the proteins, which forms two beta sheets held together by hydrophobic interactions and appears to be present in all superfamily members. The length, sequence, and amino acid composition of the N- and C-terminals, in contrast, are quite variable. The role of the N-terminal has been hypothesized to control species-specific assembly of subunits into higher level structures. To test this, a set of constructs was designed and expressed: the N-terminal sequences preceding the start of the core regions of alphaA-crystallin and HSP 16.5 from Methanococcus jannaschii were swapped; the N-terminal of each protein was removed, and replaced with a brief N-terminal extension sequence; and two nonsense N-terminal sequences of approximately the same length and hydropathicity as the original replaced the alphaA-crystallin N-terminal. All constructs, plus the original recombinant sequences, could be overexpressed except for the 16.5 N-terminal extension, and all showed chaperone-like activity except for the hybrid with the 16.5 C-terminal. Size and properties of the replacement N-terminal place limits on aggregate size. Additional restrictions are imposed by the structure of the dimer.

INSULT, a novel method for the creation of insertions, deletions, and point mutations without subcloning, requires only one new primer per mutant, and produces circular plasmids, obviating the need for special "ultracompetent" cells. The method includes cycles of linear amplification with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase. After production of multiple single-stranded copies of circular mutation-bearing plasmid DNA, addition of a "generic" primer followed by one or more polymerase reaction cycles generates double-stranded circular DNA bearing the desired mutation.

A study of bovine endothelial nitric oxide synthase by Fourier transform infrared (FTIR) spectroscopy in the 1000-2500 cm(-)(1) range is reported. Binding of CO to the reduced enzyme gives two heme(II)-CO nu(C)(-)(O) stretches (1927 and 1904 cm(-)(1)) which appear to be in rapid equilibrium. Photolysis of this heme(II)-CO compound is accompanied by perturbation of the local fine structure around the catalytic site giving vibrational changes of protein backbone, substrate, amino acid residues, and cofactors, to which heme, substrate arginine, and catalytic site residues contribute. Possible assignments of vibrations to heme, substrate arginine, and catalytic site residues are discussed. The discussion of assignments is informed by known structures, absorbance frequencies, and extinction coefficients of residues and cofactors, analysis of H(2)O-D(2)O exchange effects, analysis of substrate (14)N-(15)N (guanidinium)-arginine exchange effects, and comparison with the nNOS isoform (which differs in the replacement of asparagine 368 with an aspartate within the substrate binding site). The FTIR data can be modeled on the known structure of the catalytic site and indicate the extent of modulation of vibrational modes upon photolysis of the CO compound.

A single-stage polymerase-based procedure is described that allows extensive modifications of DNA. The version described here uses the QuikChange Site-Directed Mutagenesis System kit supplied by Stratagene. The original protocol is replaced by a single-stage method in which linear production of complementary strands is accomplished in separate single primer reactions. This has proved effective in introducing insertions and deletions into large gene/vector combinations without subcloning.

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.

The three mammalian nitric-oxide synthases produce NO from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from NADPH through reductase domains incorporating FAD and FMN cofactors. The isoforms share a common reaction mechanism and requirements for reducing equivalents but differ in regulation; the endothelial and neuronal isoforms are controlled by calcium/calmodulin modulation of the electron transfer system, while the inducible isoform binds calmodulin at all physiological Ca(2+) concentrations and is always on. The thermodynamics of electron transfer through the flavin domains in all three isoforms are basically similar. The major flavin states are FMN, FMNH., FMNH(2), FAD, FADH., and FADH(2). The FMN/FMNH. couple is high potential ( approximately 100 mV) in all three isoforms and is unlikely to be catalytically competent; the other three flavin couples form a nearly isopotential group clustered around -250 mV. Reduction of the flavins by the pyridine nucleotide couple at -325 mV is thus moderately thermodynamically favorable. The ferri/ferroheme couple in all three isoforms is approximately -270 mV in the presence of saturating arginine. Ca(2+)/calmodulin has no effect on the potentials of any of the couples in endothelial nitric-oxide synthase (eNOS) or neuronal nitric-oxide synthase (nNOS). The pH dependence of the flavin couples suggests the presence of ionizable groups coupled to the flavin redox/protonation states.

The small heat shock protein superfamily, extending over all kingdoms, is characterized by a common core domain with variable N- and C-terminal extensions. The relatively hydrophobic N-terminus plays a critical role in promoting and controlling high-order aggregation, accounting for the high degree of structural variability within the superfamily. The effects of N-terminal volume on aggregation were studied using chimeric and truncated proteins. Proteins lacking the N-terminal region did not aggregate above the tetramers, whereas larger N-termini resulted in large aggregates, consistent with the N-termini packing inside the aggregates. Variation in an extended internal loop differentiates typical prokaryotic and plant superfamily members from their animal counterparts; this implies different geometry in the dimeric building block of high-order aggregates.

The oxygenase domains of nitric oxide synthases are unusual in that they contain at least three ligand binding sites; these correspond to the axial heme ligand position, the substrate binding site, and the pterin binding site. Ligands can occupy portions of a site or extend into regions of adjacent sites. Depending on the size, shape, and binding mode of ligands to these positions, cooperative and anticooperative interactions mediated conformationally and by binding domain overlap can be observed. In the present study we describe competition between arginine and imidazole at the axial heme ligand position; a second imidazole, which occupies part of the arginine site in some crystal structures, is too weak to contribute to the equilibria. All spectroscopic titrations using imidazole competition depend on displacement of the heme axial imidazole ligand, which drives the ferriheme low spin. Aminoguanidine, a partial arginine analog, has multiple binding modes. It is somewhat competitive with arginine; a ternary complex forms, but the K(d) for arginine increases from 1 to 15 microM in the presence of saturating aminoguanidine. Aminoguanidine competition with imidazole is very weak, amounting to approximately a factor of two increase in K(d). This implies that aminoguanidine has multiple binding modes and is not well described as an arginine analog. The major binding mode occupies part of the binding site but does not extend into the imidazole axial ligand binding domain and probably corresponds to the crystal structure. The other binding mode is not significantly overlapped with the arginine site.

Objectives: To investigate the indications for, and outcome of, cardiac catheterisation in infants weighing less than 2500 g at a single institution over an 8-year period., Patients and Methods: We assessed all infants who were less than 2500 g at the time of cardiac catheterisation at Texas Children's Hospital from January 1993 to January 2001. Comparisons of morbidity and mortality were drawn with an equivalent number of infants of similar age weighing greater than 2500 g seen over the same period of time., Results: We performed interventional procedures in 22, and diagnostic catheterisations in 12 infants weighing less than 2500 g. Interventions included pulmonary valvoplasty in six patients, balloon angioplasty of critical coarctation in one, aortic valvoplasty in two, septostomy in ten, and coil occlusion of an arteriovenous malformation, redirection of a subclavian venous line, and coil occlusion of a patent arterial duct in one patient each. The median age at catheterisation was 5 days for children less than 2500 g, and 10 days for those above 2500 g. The median weights were 2.3 kg and 3.3 kg, and the median gestational ages were 35 weeks and 38 weeks, for the two respective groups. Of those weighing less than 2500 g, two died (6%), with no deaths occurring in those weighing more than 2500 g. In 3 patients weighing less than 2500 g (9%), there was vascular compromise, one child with bilateral femoral venous obstruction requiring fasciotomy compared, to one in the group weighing greater than 2500 g (2%)., Conclusion: There is a significantly increased risk of mortality and vascular compromise in infants weighing less than 2500 g. Interventional catheterisation in these infants may be lifesaving, but given the aforementioned risks, diagnostic catheterisation should be deferred if possible in favor of noninvasive modalities.

Nitric Oxide Synthases are a family of enzymes that produce NO from arginine, oxygen and reducing power in the form of NADPH; they function as signal generators and as producers of cytotoxic levels of NO (e.g., in immune defense). Evolution of eukaryotic NOS from prokaryotic antecedents involved a series of gene fusion events, producing a modular enzyme, and the concomitant development of sophisticated control mechanisms that are isoform specific and tailored to the role of enzymes in signal transduction or immune response. Recent information on the structures of NOS isoforms at all levels from primary amino acid sequence to high resolution crystallography allows a deepening understanding of many aspects of these important proteins including interdomain interactions, dimerization, cofactor, substrate, and isoform specific inhibitor binding as well catalysis and control. The details of the NOS reaction mechanism and its control through the regulation of electron transfer by CaM binding and other mechanisms are still being elucidated and are well worth further examination. The alignment of the molecular surfaces of the independently folded domains is a central feature of structure, catalysis and control in these important enzymes, and will be the focus of the present review.

Improvements in sensitivity and data processing of Fourier transform infrared (FTIR) spectroscopy enable it to be used to detect changes in protein structure at the atomic level. This paper reports a study of neuronal nitric oxide synthase (nNOS) by FTIR difference spectroscopy in the 1000-2500 cm(-1) range where vibrational bands of ligands, prosthetic groups, and protein and amino acid side chains are found. We have exploited the photolyzable CO compound of the ferrous heme of nNOS to produce light-induced CO photolysis difference spectra and to compare spectra after hydrogen/deuterium exchange. In (reduced) minus (reduced plus CO) difference spectra, negative bands at 1931 and 1907 cm(-1) are observed due to photolysis of multiple forms of ferrous heme-ligated CO, similar to those observed by resonance Raman spectroscopy [Wang et al. (1997) Biochemistry 36, 4595-4606]. Photolysis of the ferrous heme CO compound is accompanied by hitherto unreported changes in the 1000-2000 cm(-1) region that arise from changes of protein backbone, substrate, amino acid side chain, and cofactor vibrations. Preliminary assignments of vibrations are made on the basis of frequencies and the effects of hydrogen/deuterium exchange, and in the light of known atomic structures.

Spectrophotometrically monitored ligand titration is an important method for the determination of equilibrium dissociation constants (K(d)) from nitric oxide synthases (NOS). Low K(d) sites such as the tetrahydrobiopterin and arginine binding sites present difficulties in that experiments often require enzyme concentrations of the same magnitude as the K(d). An analytical method based on computer simulation is described that allows the estimation of K(d) values without an independent means of monitoring free ligand or without an accurate prior determination of the number of binding sites. The K(d) for arginine is approximately 0.5 microM for the tetrahydrobiopterin replete neuronal and inducible isoforms (nNOS and iNOS), while the endothelial isoform has a slightly higher K(d) (1.5 microM). N-OH-arginine (an intermediate) binds to nNOS with a K(d) of around 0.2 microM, while the inhibitors N-methyl-arginine and N-nitro-arginine bind more tightly; our best K(d) estimates are 100 nM or lower., (Copyright 2001 Academic Press.)

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.

It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [3H]Nomega-nitro-L-arginine or [3H]BH4, but [3H]BH4 binding was reinstated after extended incubation with excess L-arginine. On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2, 6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated Kd for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 microM versus 2-3 microM). Here we propose that Cys331 plays an important role in stabilizing L-arginine binding to nNOS. Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH4 binding and nitric oxide synthetic activity.

The quinol oxidase, cytochrome bd, functions as a terminal oxidase in the Escherichia coli respiratory chain, reducing O2 to water and using ubiquinol-8 or menaquinol-8 as its immediate reductant. The oxidation of quinol is by the low-spin ferri-haem, cytochrome b558. This occurs at a quinol-binding site by sequential one electron steps, requiring the stabilisation of the semiquinone intermediate. We have observed, by EPR spectroscopy, the properties of this semiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess of ubiquinone-8 and menaquinone-8 analogues. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a semiquinone anion of this type. The line shape is Gaussian. The semiquinone is highly stabilised with respect to free semiquinone; significant free radical can be observed at pH 7.0 and above. The pH dependence of the redox reactions indicate that the anionic form of the semiquinone and the neutral form of the quinol predominate in the pH range studied. The pH dependence of the mid-point potentials of the one electron reactions from pH 7.0-9.0 is 120 mV/pH change for the semiquinone anion to quinol (E2) and none for the quinone to semiquinone (E1). The semiquinone radical is attenuated on titration with putative inhibitors of this quinone-binding site. We have similarly studied the semiquinone in membrane preparations from a strain with overexpression of cytochrome bd oxidase. The data can be fitted with the assumption of a single quinone-binding site.

The orientation dependence of the EPR signals arising from the azide-nitric oxide complex of cytochrome oxidase was investigated using oriented multilayers of mitochondrial membranes from ox heart. Variations in line shape of the DeltaMS=1 signal of the triplet state were apparent, whilst the DeltaMS=2 transitions between g=4.7 and 3.9 varied in intensity as the angle of the applied magnetic field was varied. These half-field signals were maximal with the field parallel to the membrane plane. A model of the bi-liganded azide-nitric oxide complex has been constructed, in which the nitric oxide is bound to the high-spin haem in a bent configuration, with the Fe-N=O plane at 60-90 degrees to the membrane plane and the azide bound to the copper, distal from the haem. In addition, angular variations of the signals at g'=11 and g' around 3.5, derived from an integer-spin complex, were also observed., (Copyright 1998 Elsevier Science B.V.)

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

The binding of arginine analogs to endothelial nitric oxide synthase (eNOS, NOSIII) perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Using electron paramagnetic resonance as a probe of heme ligation geometry, four categories of high-spin complex could be distinguished. These are analogous to the four classes of high-spin complexes, stabilized individually by the binding of L-arginine, N-hydroxy-L-arginine (NHA), N-methyl-L-arginine (NMA), and N-nitro-L-arginine (NNA), which we have previously reported for the other two isoforms. Each of these species is five-coordinate and retains the axial thiolate ligand but each differs in its ligation geometry. N-Methyl-L-arginine is a relatively poor inhibitor of eNOS, and the NMA complex of eNOS differs from the N-methyl-L-arginine complexes of inducible nitric oxide synthase (iNOS, NOSII) and neuronal nitric oxide synthase (nNOS, NOSI) in that it is of lower rhombicity. We previously showed that inducible nitric oxide synthase, which binds NNA less tightly than eNOS and nNOS, could not form the lower rhombicity NNA complex characteristic of nNOS. Endothelial nitric oxide synthase readily forms such lower rhombicity complexes, which correlates with the tight binding of NNA to this isoform. Arginine and tetrahydrobiopterin promote loss of the flavin free radical EPR signal, while arginine analog inhibitors stabilize the radical; this suggests that the residual flavin radicals can serve as a source of reducing equivalents for slow turnover in the absence of endogenous reductant.

A versatile diatomic physiological messenger, nitric oxide (NO), is biosynthesized by a group of flavo-heme enzymes, the nitric oxide synthases. We have examined the active site of the neuronal isoform by EPR spectroscopy of the ferrous nitric oxide complex. The nitric oxide complex of the substrate-free enzyme exhibits a cytochrome P450-type EPR spectrum typical of a hexacoordinate NO-heme complex with a non-nitrogenous proximal axial heme ligand. The NO complex of the substrate-free enzyme is rather unstable and spontaneously converts to a cytochrome P420 type pentacoordinate denatured form. Binding of L-arginine (l-Arg) enhances the stability of the hexacoordinate NO form. The EPR spectrum of the NO adduct of the enzyme-substrate complex has an increased g-anisotropy and well-resolved hyperfine couplings due to the 14N of nitric oxide. Significant perturbations in the NO EPR spectrum were observed upon Nomega-monomethyl-L-Arg and Nomega-hydroxy-L-Arg binding. The perturbations in the EPR spectrum indicate that L-Arg and its derivatives bind on the distal site of the heme in very close proximity to the bound NO to cause alterations in the heme-NO coordination structure. Interactions between the bound NO and the substrate or its analogues appear to affect the Fe-NO geometry, resulting in the observed spectral changes. We infer that analogous interactions with oxygen might be involved in the hydroxylation events during enzyme catalysis of nitric oxide synthase.

In this article, we are interested in the problem of the existence of a few linkage groups in which lethal infertile genes associated with yield factors are present. This hypothesis is supported by the finding of natural balanced lethal systems, which keep a short heterotic chromosome segment. It is very important to predict the number of generations that a balanced lethal system maintains in its linkage, in order to get a permanent heterozygosis without any problem with recombination. For theoretical analysis we have used the theory of absorbing Markov chains, discrete in space and time, because this is well suited to crops like barley. It results from studying some experimental data obtained by Ewald A. Favret in the CICA, INTA, Castelar, Argentina.

The electron paramagnetic resonance spectra of purified neuronal nitric oxide synthase indicates that the binding of ligands to the arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Four categories of high-spin complex can be distinguished; all are five-coordinate, and all retain the axial thiolate ligand, but they differ in their ligation geometries. These spectroscopic species reveal distinct local conformations which can be stabilized individually by the binding of L-arginine, N omega-hydroxy-L-arginine, N omega-methyl-L-arginine, and N omega-nitro-L-arginine. Other arginine analog inhibitors stabilize one or more of these states, revealing patterns based on the nature of substituents at the terminal amino group.

The electron paramagnetic resonance spectra of the heme domain of inducible nitric oxide synthase (iNOS) demonstrate a close relationship to the corresponding spectra of the neuronal isoform (nNOS). The binding of ligands to the iNOS arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. The iNOS forms five-coordinate, high-spin complexes with arginine analogs which are clearly related to the corresponding complexes of nNOS. Studies indicate that the binding of L-arginine, N(omega)-hydroxy-L-arginine (NHA), and N(omega)-methyl-L-arginine (NMA) produces various spectroscopic species closely corresponding to the equivalent complexes of nNOS, while N(omega)-nitro-L-arginine (NNA) binding produces a state which appears intermediate in character between the nNOS NNA and arginine complexes. These spectroscopic studies have permitted the determination of ligand-specific high-spin states which reveal similarities and differences between iNOS and nNOS.

Nitric oxide synthase (NOS) catalyzes sequential NADPH- and O2-dependent mono-oxygenase reactions converting L-arginine to N omega-hydroxy-L-arginine and N omega-hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer, and that cofactor is thought to mediate both partial reactions. Here we show by electron paramagnetic resonance spectroscopy that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin. In contrast, binding of L-thiocitrulline, a NOS inhibitor, produces both high and low spin iron spectra; L-thiocitrulline sulfur is a sixth axial heme ligand in one, but not all, of the low spin forms. The high spin forms of the L-thiocitrulline NOS complex display a distortion in the opposite direction to that caused by L-arginine binding. The findings elucidate the binding interactions of L-arginine and L-thiocitrulline to neuronal NOS and demonstrate that each causes a unique perturbation to the heme cofactor pocket of NOS.

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome-c-binding site and its attendant CuA prosthetic group with a quinone-binding site. The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one-electron steps requires the stabilisation of semiquinone. We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone. The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5-10. The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5. The two-electron couple has an Em7 of approximately 70 mV. Below pH 9, the pH dependence of the two-electron couple is -60mV/pH, indicative of a 2H+/2e- reaction. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q.- species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q.- radicals. Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q.-bo (QH.bo).

Starch biogenesis in corn endosperm from Flint, Sugary, Waxy, as a function of the grain filling/period was studied. We have differentially identified the initiation from the elongation process. After incubating under unprimed conditions, two glucose radiolabelled protein bands of 39,5 and 36 kDa were obtained. UDP(14C)Glc was the preferred glucosyl donor but also ADP(14C)Glc was. It was additionally found that more than one glucose was transferred to the protein or to the alpha 1,4-glucan linked to protein from UDPGlc. These results were supported by the fact that the glucosylated protein from UDPGlc liberates maltooligosaccharides after alpha- or beta-amylase treatment. The elongation activity in the first steps related to the glucan linked to protein is different from starch synthase. Therefore, we are proposing a model for starch biogenesis where two new transglucosylating enzyme activities are necessary to prepare the primer for starch synthase.

The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa3-type cytochrome c oxidase. The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a3) and copper (CuB). Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo. Cyanide, sulfide, and azide react with the Fe(3+)-Cu+ center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa3. Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand. Cyanide and azide react with the Fe(3+)-Cu2+ center to yield forms observable via UV-visible spectroscopy but not EPR. With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa3. These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet. Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa3. This suggests that the binuclear centers of the two enzymes are quite similar.