[Structure-function relationships of NO synthase: electron transfer reaction].
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Animals↗
Biomedical subjects
Toru Shimizu
Publications and source records attributed to Toru Shimizu.
Unusual cyanide bindings to a heme-regulated phosphodiesterase from Escherichia coli: effect of Met95 mutations.
In order to understand heme environment of a heme-regulated phosphodiesterase (Ec DOS), the binding behavior of cyanide to the Fe (III) complex was examined. Interestingly, the rate of cyanide binding to full-length Ec DOS was unusually slow with k(on)=0.0022mM(-1)s(-1), while the rate for the isolated heme domain of Ec DOS (0.045mM(-1)s(-1)) was 20-fold higher. Ala and Leu mutations at Met95, which has been suggested to be a heme axial ligand, increased the k(on) rate 11- and 8-fold, respectively, and dramatically decreased the cyanide dissociation rate from the isolated heme domain. His mutation at Met95, on the other hand, caused a 17-fold decrease in the k(on) value. We discuss the unusual cyanide binding behavior and the role of Met95 in controlling cyanide binding.
Azides↗
Catalytically functional flavocytochrome chimeras of P450 BM3 and nitric oxide synthase.
P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains. Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras. Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified. All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity. However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case. The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties.
Animals↗
Stationary and time-resolved resonance Raman spectra of His77 and Met95 mutants of the isolated heme domain of a direct oxygen sensor from Escherichia coli.
The heme environments of Met(95) and His(77) mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band (nu(Fe-His)) at 214 cm(-1), and the nu(Fe-CO) versus nu(CO) plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the nu(Fe-His) band but gave a nu(Fe-Im) band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a five-coordinate heme complex. Taken together, we suggest that the heme of the reduced WT enzyme is coordinated by His(77) and Met(95), and that Met(95) is displaced by CO and O(2). Presumably, the protein conformational change that occurs upon exchange of an unknown ligand for Met(95) following heme reduction may lead to activation of the phosphodiesterase domain of E. coli DOS.
Carbon Monoxide↗
Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.
A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its alpha-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min(-1) with cAMP, which was optimal at pH 8.5 in the presence of Mg(2+) and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.
3',5'-Cyclic-AMP Phosphodiesterases↗
Interactions between the isolated oxygenase and reductase domains of neuronal nitric-oxide synthase: assessing the role of calmodulin.
Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca(2+)/calmodulin (CaM) for NO formation activity. We studied NO formation activity in reconstituted systems consisting of the isolated oxygenase and reductase domains of neuronal NOS with and without the CaM binding site. Reductase domains with 33-amino acid C-terminal truncations were also examined. These were shown to have faster cytochrome c reduction rates in the absence of CaM. N(G)-hydroxy-l-Arg, an intermediate in the physiological NO synthesis reaction, was found to be a viable substrate. Turnover rates for N(G)-hydroxy-l-Arg in the absence of Ca(2+)/CaM in most of the reconstituted systems were 2.3-3.1 min(-1). Surprisingly, the NO formation activities with CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca(2+)/CaM. However, NADPH oxidation and cytochrome c reduction rates were increased by the same procedure. Activation of the reductase domains by CaM addition or by C-terminal deletion failed to increase the rate of NO synthesis. Therefore, both mechanisms appear to be less important than the domain-domain interaction, which is controlled by CaM binding in wild-type neuronal NOS, but not in the reconstituted systems.
Animals↗
Significance of visual cues in choice behavior in the female zebra finch (Taeniopygia guttata castanotis).
Female zebra finches show a preference for male zebra finches over heterospecific males based solely on the auditory cues of males, such as songs. The present study was designed to investigate whether females show a similar preference for male zebra finches based solely on visual cues. Using a Y-maze apparatus, social preference of female zebra finches was studied between male zebra finches and male Bengalese finches in three experiments. In experiment 1, where female zebra finches could see and hear live male zebra finches and male Bengalese finches, the females preferred to associate with the male zebra finches. In experiment 2, using a sound-attenuated experimental apparatus, subjects could see, but not hear, male zebra finches and male Bengalese finches. The subjects did not show a significant preference for associating with zebra finches. In experiment 3, as in experiment 2, females could see live male zebra finches and male Bengalese finches in the sound-attenuated chambers. However, in experiment 3, the subjects also heard prerecorded auditory cues (i.e., songs and calls) of male zebra finches, which were presented simultaneously in both arms of the maze. Although the females could not use the auditory cues to identify the location of the male zebra finches, they preferred to associate with the male zebra finches rather than the male Bengalese finches. These results suggest that visual cues alone were effective in initiating choice behaviors by females and that auditory cues facilitate such visually based choice behaviors.
Animals↗
Inhibition of inducible nitric oxide synthase attenuates interleukin-1 beta induced vascular hyporeactivity in the rabbit.
Inhibition of nitric oxide (NO) synthesis has been indicated to improve vasopressor-responsiveness and to increase blood pressure in most septic models. However, numerous adverse effects of non-selective NO synthase (NOS) inhibition have been reported, and the effect of NOS inhibition on vascular responsiveness to vasodilators has not been well studied. Using an isometric tension measurement system of vascular rings, we evaluated the effects of an inducible NOS (iNOS) inhibitor, L-canavanine (L-CAN) and a non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on interleukin-1 beta (IL-1 beta)-induced vascular hyporeactivity in the four different rabbit arteries. Pretreatment of IL-1 beta inhibited phenylephrine (Phe)-induced vascular constriction in the carotid artery (CA, 49% of control), pulmonary artery (PA, 66%), femoral artery (FA, 71%) and in the renal artery (RA, 83%). A combination of NOS inhibitors attenuated the vascular hyporeactivity to Phe in all arteries. Pretreatment of IL-1 beta also inhibited acetylcholine (Ach)-induced vascular relaxation in FA, RA and CA. In PA, the rings were inversely constricted after Ach administration. The combination of IL-1 beta with L-CAN, but not with L-NAME, attenuated the Ach-induced vasorelaxation to the control level in all arteries. These data suggest that the selective inhibition of iNOS attenuates the direct endothelial damage induced by IL-1 beta in vitro.
Animals↗