Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen.

The heme domain (iNOS(heme)) of inducible nitric oxide synthase (NOS) was expressed in Escherichia coli and purified to homogeneity. Rapid freeze-quench (RFQ) EPR was used to monitor the reaction of the reduced iNOS(heme) with oxygen in the presence and absence of substrate. In these reactions, heme oxidation occurs at a rate of approximately 15 s(-)(1) at 4 degrees C. A transient species with a g = 2.0 EPR signal is also observed under these conditions. The spectral properties of the g = 2.0 signal are those of an anisotropic organic radical with S = (1)/(2). Comparison of the EPR spectra obtained when iNOS(heme) is reconstituted with N5-(14)N- and (15)N-substituted tetrahydrobiopterin (H(4)B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H(3)B.) of the bound H(4)B. Substitution of D(2)O for H(2)O reveals the presence of hyperfine-coupled exchangeable protons in the H(4)B radical. This radical forms at a rate of 15-20 s(-)(1), with a slower decay rate that varies (0.12-0.7 s(-)(1)) depending on the substrate. At 127 ms, H(3)B. accumulates to a maximum of 80% of the total iNOS(heme) concentration in the presence of arginine but only to approximately 2.8% in the presence of NHA. Double-mixing RFQ experiments, where NHA is added after the formation of H(3)B., show that NHA does not react rapidly with H(3)B. and suggest that NHA instead prevents the formation of the H(4)B radical. These data constitute the first direct evidence for an NOS-bound H(3)B. and are most consistent with a role for H(4)B in electron transfer in the NOS reaction.

Catalysis by nitric oxide synthase.

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.

Computer-mediated social support: single young mothers as a model system.

Forty-two single mothers with young infants were given access to a computer-mediated social support (CMSS) network concerned with parenting issues. The network operated 24 hours per day over a period of 6 months. It permitted public message exchanges, private e-mail, and text-based teleconferencing for as many as 8 participants at any one time. During the 6 month intervention, the 42 women accessed the network over 16,670 times. Individual differences in participation were significantly associated with indices of social isolation from peers. A descriptive analyses of the messages exchanged on the network disclosed that 98% of the replies to concerns posted in the public forum provided positive social support. The majority of the supportive replies fell into the category of emotional support, followed in order by informational and tangible support. Both the self-report data following the intervention, and qualitative data extracted from online discussions indicated that close personal relationships and a sense of community developed in this novel social environment. Finally, an analysis of pretest-posttest changes in the level of parenting stress revealed that mothers who participated regularly in this CMSS community were more likely to report a decrease in parenting stress following the intervention.

Nitric oxide complexes of inducible nitric oxide synthase: spectral characterization and effect on catalytic activity.

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (.NO). NOS is a hemoprotein containing a cytochrome P-450-type heme that has been shown to be involved in catalysis. It has been suggested that .NO is able to bind tightly to the heme of NOS and may in this way serve to regulate enzymatic activity. We report here the formation of both ferric and ferrous heme nitrosyl complexes with the inducible NOS from murine macrophages. The ferric nitrosyl complex is characterized by a Soret peak at 443 nm and two distinct peaks in the alpha/beta region at 549 and 585 nm. The ferrous nitrosyl complex has absorbance maxima at 436 and 566 nm. A transient spectral intermediate is observed under conditions of NOS turnover. This intermediate appears to be a mixture of ferric and ferrous nitrosyl complexes and is unstable in the presence of oxygen. Binding of L-arginine decreases the affinity of .NO for the ferric heme but does not appear to decrease the affinity of .NO for the ferrous heme. Addition of either oxyhemoglobin or methemoglobin to NOS assays results in a nearly 2-fold increase in enzymatic activity. This result is attributed to the ability of both forms of hemoglobin to decrease the concentration of .NO in solution and is consistent with .NO inhibition of NOS under assay conditions. Our results show that NOS nitrosyl complexes form under certain conditions but suggest that the relevance of such complexes to activity in vivo may be limited by their instability in an aerobic environment.

Social contingency effects on subsequent perceptual-cognitive tasks in young infants.

3 experiments with 3-month-old infants compared the effects of contingent and noncontingent adult-infant social interactions on subsequent infant-controlled habituation and choice tasks. Infants who experienced a prior noncontingent social interaction tended to adopt response strategies that reduced the density of stimulation during these subsequent nonsocial tasks. The results are discussed in terms of their generality and the types of mechanisms that might mediate these transfer effects from social to nonsocial procedures.