Reflection of the plastic properties of very simple neuronal assemblies in the statistical characteristics of the spike activity of their elements. Monosynaptically connected neurons

Changes of cross-correlation histograms (CCH) of impulse trains and of mean interspike intervals (ISI) of monosynaptically connected neurones under changes of interneuronal connection efficiency, neuronal excitability and action on these neurones of independent irregular afferent synaptic inflows, were studied by methods of mathematical and biomathematical modelling of synaptic neuronal interaction (computer-controlled experiment on mollusc neurones). It was shown that statistical of increase of efficiency of monosynaptic excitatory or inhibitory interneuronal connection (amplitude enhancement of corresponding postsynaptic potential) is an increase of the main peak or trough of normalized CCH of impulse trains accompanied by a decrease of mean ISIs of both neurones.     

The analysis of the impulse activity of 2 neurons monosynaptically excitable by a 3rd using cross-correlation histograms and Cox's method. The potential for detecting neuronal and synaptic plasticity

By a method of mathematical modelling exogenic stationary random single impulse activity was reproduced of two neurones (N1 and N2), monosynaptically excited by a third one (N3). Value P12, defined from cross-correlation histogram and Cox coefficient beta 12 were used to evaluate the degree of dependence of N1 and N2 impulse trains. Dynamics of P12, beta 12 and of values of P1*, P2* and P3* proportional to corresponding mean interimpulse intervals of N1, N2 and N3, was studied under changes of efficiency of interneuronal connections, neurones excitability and summate action on them of independent random afferent synaptic inflows. It has been shown that the increase (decrease) of P12 or beta 12 accompanied by decrease (increase) of P1* and P2* is a sign of plastic change or N3 discharges frequency, or a sign of plastic changes of the amplitude of excited postsynaptic potentials elicited by these discharges.     

Reflection of the plastic properties of monosynaptically interconnected neurons in the statistical characteristics of their spike activity

By mathematical and biomathematical methods of neuronal interaction modelling, changes were studied of cross-correlation histograms (CCH) of impulse flows and of average interimpulse intervals of monosynaptically interconnected neurones, at changes of efficiency of forward and backward connections, of excitability of neurones and of summate action on them of independent random afferent synaptic inflows. It is shown, that a single sign of efficiency increase of monosynaptic excitatory or inhibitory connection between neurones (amplitude increase of the corresponding postsynaptic potential) consists in amplitude increase of the main peak or trough the rated CCH of their impulse flows, followed by a decrease of average interspike intervals of both neurones.     

Molecular cloning in yeast by in vivo homologous recombination of the yeast putative alpha1 subunit of the voltage-gated calcium channel

Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ channel pore-forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli. Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli. Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch-activated Ca2+ channel component, does not increase Ca2+ uptake activity, but co-overexpression results in a 2- to 3-fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co-overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity.     

2D NMR study of the DNA duplex d(CTCTC*A*ACTTCC).d(GGAAGTTGAGAG) cross-linked by the antitumor-active dirhodium(II,II) unit at the cytosine-adenine step

The 2D NMR analysis in solution of the DNA duplex d(CTCTC*A*ACTTCC).d(GGAAGTTGAGAG) binding to the dirhodium unit cis-[Rh2(mu-O2CCH3)2(eta1-O2CCH3)]+ showed that an unprecedented intrastrand adduct, dsII, is formed with the dirhodium unit cross-linking in the major groove residues C5 and A6 (indicated with asterisks), also corroborated by enzyme digestion studies. Formation of the dirhodium complex dsII destabilizes significantly the duplex as indicated by the substantial decrease in its melting temperature (DeltaTm = -22.9 degrees C). The reduced thermal stability of dsII is attributed to the decreased stacking of the bases and the complete disruption and/or weakening of the hydrogen bonds within the base pairs in the immediate vicinity of the metalation site (C5.G20 and A6.T19), but the effects due to the metal binding are more severe for the base pairs in the 5' direction to the lesion site. The NMR spectroscopic data indicate that Watson-Crick hydrogen bonding is completely disrupted for the C5.G20 site and considerably weakened for A6.T19. In dsII, the bases C5 and A6 bind to eq positions of the dirhodium unit cis-[Rh2(mu-O2CCH3)2(eta1-O2CCH3)]+, which retains one monodentate and two bridging acetate groups, presumably due to steric reasons. Binding of A6 takes place via N7, whereas binding of the C5 base takes place via the exocyclic N4 site, resulting in the anti-cytosine rotamer with respect to site N3 in its metal-stabilized rare iminooxo form.     

Recovery functions of somatosensory vertex potentials in man: interaction of evoked responses from right and left fingers

Somatosensory vertex potentials (SVPs) were examined in 12 healthy subjects in response to painful electrical stimulation of the finger. SVPs consisted of N1, P1, and N2. The average latencies of the 3 peaks were 150, 225, and 350 msec, respectively. The latency and amplitude of each potential were reproducible for each subject. Recovery functions of the SVPs were analyzed in 10 subjects. A pair of stimuli were delivered to the right or left finger with interstimulus intervals (ISIs) of 50, 100, 150, 200, 350, 500 and 650 msec. SVPs partially recovered with the shortest ISI (50 msec). Full recovery could not be obtained even with the longest ISI (650 msec). Differences in recoveries within 650 msec of ISI were not observed between right and left stimulations. To examine the interaction between SVPs evoked by right and left finger stimulation, recovery functions from prior contralateral finger stimulation were analyzed with the same ISIs. SVP recoveries for right after left or left after right patterns of stimulus delivery were nearly the same as those for ipsilateral ones. It is suggested that SVPs are generated at nearly the same site in the sensory pathway regardless of the side stimulated.     

Effects of interstimulus interval on somatosensory evoked magnetic fields (SEFs): a hypothesis concerning SEF generation at the primary sensorimotor cortex

Cerebral responses evoked by peripheral stimuli are known to depend critically on the interstimulus interval (ISI). Here we report on the effects of ISI on somatosensory evoked magnetic fields (SEFs) to right median nerve stimulation, obtained in 9 healthy adults with ISIs of 0.15, 0.3, 1, 3 and 5 s. At the contralateral (left) primary sensorimotor cortex (SMI), the first cortical response, N20m, was stable between the ISIs 0.3 and 5 s, but slightly attenuated at the shortest ISI of 0.15 s. In contrast, the P35m and P60m deflections were very sensitive to changes of the ISI, declining steadily with shortening of the ISI throughout the entire range. These deflections were frequently undetectable at the shortest ISI of 0.15 s. Concomitant with the reductions of P35m and P60m, an N45m deflection was enhanced toward the short ISIs. Responses from second somatosensory cortex (SII) and posterior parietal cortex (PPC) were seen only with ISIs of 1 s or greater, being strongest at the 5 s ISI. Based on known effects of the ISI on intracellular evoked potentials, we present the following tentative model for the generation mechanism of the SMI response: N20m represents early excitatory postsynaptic potentials (EPSPs), P35m early inhibitory postsynaptic potentials (IPSPs), N45m secondary EPSPs and P60m late IPSPs in pyramidal neurones of area 3b. For practical purposes, SEFs from SMI can be obtained with short ISIs, while responses from SII and PPC require an ISI of at least 1 s.     

Effects of aging on event-related brain potentials (ERPs) in a visual detection task

Event-related brain potentials (ERPs) were recorded from 74 subjects (45 men) between 18 and 82 years of age in a simple visual detection task. On each trial the subject reported the location of a triangular flash of light presented briefly 20° laterally to the left or right visual field or to both fields simultaneously. ERPs to targets exhibited a similar morphology including P1, N1, P2, N2, and P3 components across all age groups. The principal effects of advancing age were (1) a marked reduction in amplitude of the posterior P1 component (75–150 latency) together with an amplitude increase of an anterior positivity at the same latency; (2) an increase in amplitude of the P3 component that was most prominent over frontal scalp areas; and (3) a linear increase in P3 peak latency. These results extend the findings of age-related changes in P3 peak latency and distribution to a non-oddball task in the visual modality and raise the possibility that short-latency ERPs may index changes in visual attention in the elderly.     

A comparative study of the thermal stability of oligodeoxyribonucleotides containing 5-substituted 2'-deoxyuridines.

Two series of modified oligonucleotides based on the self-complementary dodecamer d(CGCTAATTAGCG) were synthesized. The first contained the -C identical withCCH2R linker at C5 of deoxyuridine at position 4 (T*) of d(CGCT*AATTAGCG) and the second contained the -SR linker. The goal of the study was to evaluate and compare these two types of side chains for suitability as tethers for linking reporter groups to oligonucleotides. Our primary concern was how these tethers would effect duplex stability. The modified nucleosides were synthesized by palladium-mediated coupling reactions between the substituted alkyne and 5'-(4, 4'-dimethoxytrityl)-5-iodo-2'-deoxyuridine and between a disulfide and 5-chloromercurio-2'-deoxyuridine. The C5 deoxyuridine side chains evaluated included C identical with CCH3, C identical with CCH2NHC(O)CH3, C identical with CCH2N(CH3)2, C identical with CCH2N-HC(O)C5H4N, C identical with CCH2NHC(O)C10H15, SCH3, SC6H5 and SCH2CH2NHC(O)CH3. The nucleosides containing these substituents were incorporated into oligo-deoxyribonucleotides by standard phosphoramidite methodology. Melting studies demonstrated that the sequence containing the C identical with CCH3side chain had the highest T m value (59.1 degrees C) in comparison with the control sequence (T m = 55.2 degrees C) and that any additional substituent on C3 of the propynyl group lowered the T m value relative to propynyl. Nevertheless, even the most destabilizing substituent, adamantylcarbamoyl, yielded an oligodeoxyribonucleotide that dissociated with a T m of 54 degrees C, which is only 1.2 degrees C less than the control sequence. In contrast, the thioether substituents led to lower T m values, ranging from as low as 45.1 degrees C for SPh up to 52.2 degrees C for SMe. Replacing the methyl of the SMe substituent with a CH2CH2NHC(O)CH3 tether led to no further reduction in melting temperature. The T m value of the CH2CH2NHC(O)CH3-containing oligonucleotide was less than the natural sequence by 1.6 degrees C/substituent. This is sufficiently small that it is anticipated that the C5 thioether linkage may be as useful as the acetylenic linkage for tethering reporter groups to oligonucleotides. More importantly, the thioether linkage provides a means to position functional groups to interact specifically with opposing complementary (target) sequences.     

The Enhancement of the N1 Wave Elicited by Sensory Stimuli Presented at Very Short Inter-Stimulus Intervals Is a General Feature across Sensory Systems

Background

A paradoxical enhancement of the magnitude of the N1 wave of the auditory event-related potential (ERP) has been described when auditory stimuli are presented at very short (<400 ms) inter-stimulus intervals (ISI). Here, we examined whether this enhancement is specific for the auditory system, or whether it also affects ERPs elicited by stimuli belonging to other sensory modalities.

Methodology and Principal Findings

We recorded ERPs elicited by auditory and somatosensory stimuli in 13 healthy subjects. For each sensory modality, 4800 stimuli were presented. Auditory stimuli consisted in brief tones presented binaurally, and somatosensory stimuli consisted in constant-current electrical pulses applied to the right median nerve. Stimuli were delivered continuously, and the ISI was varied randomly between 100 and 1000 ms. We found that the ISI had a similar effect on both auditory and somatosensory ERPs. In both sensory modalities, ISI had an opposite effect on the magnitude of the N1 and P2 waves: the magnitude of the auditory and the somatosensory N1 was significantly increased at ISI≤200 ms, while the magnitude of the auditory and the somatosensory P2 was significantly decreased at ISI≤200 ms.

Conclusion and Significance

The observation that both the auditory and the somatosensory N1 are enhanced at short ISIs indicates that this phenomenon reflects a physiological property that is common across sensory systems, rather than, as previously suggested, unique for the auditory system. Two of the hypotheses most frequently put forward to explain this observation, namely (i) the decreased contribution of inhibitory postsynaptic potentials to the recorded scalp ERPs and (ii) the decreased contribution of ‘latent inhibition’, are discussed. Because neither of these two hypotheses can satisfactorily account for the concomitant reduction of the auditory and the somatosensory P2, we propose a third, novel hypothesis, consisting in the modulation of a single neural component contributing to both the N1 and the P2 waves.     

Incorporation of Chitosan Microspheres into Collagen-Chitosan Scaffolds for the Controlled Release of Nerve Growth Factor

Background

Artifical nerve scaffold can be used as a promising alternative to autologous nerve grafts to enhance the repair of peripheral nerve defects. However, current nerve scaffolds lack efficient microstructure and neurotrophic support.

Methods

Microsphere–Scaffold composite was developed by incorporating chitosan microspheres loaded with nerve growth factor (NGF–CMSs) into collagen-chitosan scaffolds (CCH) with longitudinally oriented microchannels (NGF–CMSs/CCH). The morphological characterizations, in vitro release kinetics study, neurite outgrowth assay, and bioactivity assay were evaluated. After that, a 15-mm-long sciatic nerve gap in rats was bridged by the NGF–CMSs/CCH, CCH physically absorbed NGF (NGF/CCH), CCH or nerve autograft. 16 weeks after implantation, electrophysiology, fluoro-gold retrograde tracing, and nerve morphometry were performed.

Results

The NGF–CMSs were evenly distributed throughout the longitudinally oriented microchannels of the scaffold. The NGF–CMSs/CCH was capable of sustained release of bioactive NGF within 28 days as compared with others in vitro. In vivo animal study demonstrated that the outcomes of NGF–CMSs/CCH were better than those of NGF/CCH or CCH.

Conclusion

Our findings suggest that incorporation of NGF–CMSs into the CCH may be a promising tool in the repair of peripheral nerve defects.     

The influence of stimulus intensity and inter-stimulus interval on the detection of pitch and loudness changes

This study illuminates processes underlying change detection for different features (detection of pitch versus loudness changes) and different amounts of attentional allocation (automatic versus attentive change detection). For this reason, the influence of important stimulus characteristics (intensity and inter-stimulus interval (ISI)) on these different types of change detection was determined. By varying intensity, it should be clarified whether these processes are mainly sensitive to the informational content of the change or to the total amount of stimulus energy. By varying ISI, it should be determined whether they are differentially sensitive to manipulations of encoding time and/or state of sensory refractoriness. Automatic change detection was indexed by the mismatch negativity (MMN), which is a component of the event-related brain potential (ERP). Attentive change detection was indexed by the N2b and P3 components of the ERP and by behavioral performance. Human subjects were presented with a high-probability standard tone and a low-probability deviant-tone, which differed from the standard tone in frequency (Experiment I) or intensity (Experiment II). In separate blocks, the intensities of the standard stimuli were of 55 and 70 dB SPL and ISIs were of 350 and 950 ms. During the first part of the experiments, subjects were engaged in silent reading, whereas they tried to discriminate deviants from standards in the second part. The MMN elicited by a frequency change was invariant to variations in intensity and ISI, whereas the MMN elicited by an intensity change was significantly modulated by both intensity and ISI. This implies functional differences between the neural traces underlying the frequency-MMN and the intensity-MMN. In addition, there were larger effects of the ISI on the N2b and P3 amplitudes as compared with the effects on the MMN amplitudes, suggesting stronger capacity limitations for attentive change detection than for automatic change detection.     

Effects of ovine follicular fluid on plasma LH and FSH secretion in ovariectomized ewes to indicate the site of action of inhibin

Ovariectomized ewes were given 2 ml s.c. injections of ovine follicular fluid (oFF) (N = 3) or serum (N = 3) and blood samples were collected each day for 3 days. Follicular fluid caused a significant (P less than 0.005) reduction in FSH within 1 day, but did not affect mean LH values. Two groups of 3 ewes were treated as above but sampled intensively (each 10 min for 6 h) on Days 1 (before treatment) and 4; mean plasma FSH concentration and plasma LH pulse frequency and amplitude were ascertained. Significant (P less than 0.005) reduction of FSH concentration was seen in the oFF-treated ewes. A non-specific reduction in LH pulse amplitude, but not pulse frequency, was noted in the control ewes. This experiment was repeated with 2 groups of 4 ewes that were conditioned to the experimental environment and effects on LH secretion were not observed in the controls given serum. Treatment with oFF caused a 70% reduction (P less than 0.005) in plasma FSH and a small (30%) but significant (P less than 0.005) reduction in mean LH concentrations. The latter was probably associated with a reduction in LH pulse amplitude in 3/4 animals (N.S.) with no change in LH pulse frequency. Treatment with oFF, as in Exp. 1, caused a 95% reduction in FSH values and significant (P less than 0.01) reduction (32%) of LH pulse amplitude in ovariectomized ewes that had been subjected to hypothalamo-pituitary disconnection and in which gonadotrophin secretion was reinstated with pulses of 250 ng GnRH every 2 h. These results suggest that proteins from the sheep follicular fluid, including inhibin, act at the pituitary level to inhibit FSH secretion and may have some effects on LH pulse amplitude.     

Single-trial latency variability does not contribute to fast habituation of the long-latency averaged auditory evoked potential in the albino rat

Fas habituation (FH) is defined as a general reduction in long-latency, vertex-recorded, averaged auditory evoked potential (AEP) amplitude that occurs in response to the second of a pair of acoustic stimuli. Our laboratory has been studying FH in a variety of human populations with different paradigms and has interpreted it to be a measure of neural attentional mechanism(s) and/or resource allocation related to the processing of cognitive information. We have also reported an analogous phenomenon in the rat. In the present investigation, we examined the relationship between FH (viz., averaged AEP component amplitude decrement) and the single-trial latency variability of the AEP peaks comprising that component. Specifically, AEPs were obtained to 60 paired-tone stimuli from unanesthetized and restrained albino rats previously implanted with chronic skull electrodes. Using a template-matching algorithm similar to that used by Michalewski et al. (Electroenceph. clin. Neurophysiol., 1986, 65:59–71), the latency variability for each animal was computed for the N1 and P2 peaks of the single-trial AEPs that were used to compose the averaged wave form. Findings indicated that (a) there was no difference in single-trial latency variability for these peaks either within or across tones, and (b) there was no relationship between single-trial latency variability for either the N1 or the P2 peaks and the overall peak-to-peak amplitude (N1-P2) of the averaged wave form in response to the second tone. Thus, FH of the N1-P2 (i.e. Peak 2) amplitude in the rat is not due to an increase in latency variability across tones.     

The intelligibility of noise-vocoded speech: spectral information available from across-channel comparison of amplitude envelopes

Noise-vocoded (NV) speech is often regarded as conveying phonetic information primarily through temporal-envelope cues rather than spectral cues. However, listeners may infer the formant frequencies in the vocal-tract output-a key source of phonetic detail-from across-band differences in amplitude when speech is processed through a small number of channels. The potential utility of this spectral information was assessed for NV speech created by filtering sentences into six frequency bands, and using the amplitude envelope of each band (≤30 Hz) to modulate a matched noise-band carrier (N). Bands were paired, corresponding to F1 (≈N1 + N2), F2 (≈N3 + N4) and the higher formants (F3' ≈ N5 + N6), such that the frequency contour of each formant was implied by variations in relative amplitude between bands within the corresponding pair. Three-formant analogues (F0 = 150 Hz) of the NV stimuli were synthesized using frame-by-frame reconstruction of the frequency and amplitude of each formant. These analogues were less intelligible than the NV stimuli or analogues created using contours extracted from spectrograms of the original sentences, but more intelligible than when the frequency contours were replaced with constant (mean) values. Across-band comparisons of amplitude envelopes in NV speech can provide phonetically important information about the frequency contours of the underlying formants.     

C-myc expression in the microvessels of medulloblastoma

The increased expression of c-myc is related to neoplastic transformation and angiogenesis. Therefore, the assessment of expression of c-myc in endothelial cells and neovascularization could help to determine the biological behavior of the tumor. We analyzed neovascularization and c-myc expression in 36 medulloblastoma specimens. The results were shown by determining immunohistochemical staining index (ISI), the sum of staining intensity (SI) and the percentage of positive cells (PPC) in the blood vessels endothelium of the tumor. We also performed the microvessel count (MVC) in 10 high-power fields (400X) with the most prominent vascularization and expressed it as microvessel density per mm2 (MVD). C-myc immunostaining intensity index in blood vessel endothelium is grouped into four groups, 0--no reaction, I-weak reaction (ISI = 1 or 2), II--moderate reaction (ISI = 3 or 4), III--strong reaction (ISI = 5 or 6). Statistically significant differences (p = 0.0214) have been found between groups 0 and 1 compared to groups 2 and 3. A higher percentage of positive cells has been found in male patients than in female ones (p = 0.0483). C-myc PPC 0 or 1 has on the average smaller density of blood vessels per mm2 than c-myc PPC 2 or 3, but the difference is not statistically significant. C-myc ISI 0 or 1 has, on the average, smaller density of blood vessels per mm2 than c-myc ISI 2 or 3, but the difference is not statistically significant. We concluded that c-myc staining intensity was associated with higher microvessels density.     

Receptor density determines secretory response patterns mediate by inositol lipid-derived second messengers. Comparison of thyrotropin-releasing hormone and carbamylcholine actions in thyroid-stimulating hormone-secreting mouse pituitary tumor cells

Signal transduction by thyrotropin-releasing hormone (TRH) and carbamylcholine (CCH) in some cells is mediated by inositol lipid hydrolysis forming the second messengers, inositol 1,4,5-trisphosphate (I-1,4,5-P3) and 1,2-diacylglycerol, and causing elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i). In mouse thyrotropic tumor (TtT) cells, maximally effective doses of TRH caused biphasic stimulation of thyroid-stimulating hormone (TSH) secretion, whereas CCH stimulated monophasic sustained TSH secretion without a burst phase. TRH, at maximally effective doses, stimulated a rapid marked increase in I-1,4,5-P3 which was associated with a rapid elevation of [Ca2+]i to approximately 1000 nM, whereas maximally effective doses of CCH caused little increase in I-1,4,5-P3 and no burst elevation of [Ca2+]i. Both TRH and CCH caused sustained modest (to 210-280 nM) elevations of [Ca2+]i which were inhibited by voltage-sensitive channel-blocking agents and stimulated sustained hydrolysis of inositol lipids. CCH-like responses were observed when TtT cells were stimulated by low doses of TRH. In TtT cells prepared from five tumors, the ratio of the number of TRH receptors to muscarinic receptors ranged from 10 to 40:1. Lastly, CCH-like responses were observed with maximally effective doses of TRH when the TRH receptor number was down-regulated to a level similar to that of muscarinic receptors. These data suggest that the kinetic pattern of stimulated TSH secretion caused by secretagogues that use the inositol lipid signal transduction pathway is determined by the density of receptors. In particular, there appears to be a minimal number of receptor-ligand complexes which is required to generate rapidly sufficient I-1,4,5-P3 to release intracellular Ca2+ and cause a secretory burst.     

Fast habituation of the long-latency auditory evoked potential in the awake albino rat

Fast habituation of the long-latency, vertex-recorded auditory evoked potential (AEP) peaks in humans was first described by Callaway (1973) as a reduction in AEP amplitude that occurs to the second of a pair of acoustic stimuli when both stimuli are presented with an interstimulus interval (ISI) of no more than 10 sec. When acoustic stimuli are presented in pairs with an ISI of 2 sec and an interpair interval (IPI) of approximately 10 sec, reduction in amplitude to the second tone occurs by as much as 30–50%. Fast habituation may depend somewhat on a subject's anticipation of the stimulus and on other factors related to attention and orienting. Studies in our laboratory have demonstrated this amplitude decrement to the second tone of a pair in human infants, children and adults and have explored the implications of this finding with respect to attentional processes and the allocation of cerebral resources. In the present investigation we describe an animal model of fast habituation. Here, vertex-recorded AEPs were obtained to paired tone stimuli delivered to awake adult male Sprague-Dawley rats chronically implanted with skull electrodes. Findings showed: (a) an AEP wave form with 8 distinct peaks, (b) for one component there was a marked decrement in amplitude from tone 1 to tone 2 in recordings obtained from an electrode placed slightly to the right of midline, and (c) that there were no significant differences in peak latencies across tones. This methodology may further our understanding of fast habituation in humans and may prove useful for studies of attention, orienting, and resource allocation using techniques that are not possible for use with human subjects.     

Localization of a catalytic intermediate bound to the FeMo-cofactor of nitrogenase

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HC identical with CH) to ethylene (H2C=CH2). It is known that the metallocluster FeMo-cofactor located within the nitrogenase MoFe protein component provides the site of substrate reduction, but the exact site where substrates bind and are reduced on the FeMo-cofactor remains unknown. We have recently shown that the alpha-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; alpha-70 approaches one face of the FeMo-cofactor, and when valine is substituted by alanine at this position, the substituted nitrogenase is able to accommodate a reduction of the larger alkyne propargyl alcohol (HC identical with CCH(2)OH, propargyl-OH). During this reduction, a substrate-derived intermediate can be trapped on the FeMo-cofactor resulting in an S = 1/2 spin system with a novel electron paramagnetic resonance spectrum. In the present work, trapping of the propargyl-OH-derived or propargyl amine (HC identical with CCH(2)NH(2), propargyl-NH(2))-derived intermediates is shown to be dependent on pH and the presence of histidine at position alpha-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the-OH group or the-NH(3)(+)group and the imidazole epsilon-NH of alpha-195(His). Thus, for the first time it is possible to establish the location of a bound substrate-derived intermediate on the FeMo-cofactor. Refinement of the binding mode and site was accomplished by the use of density functional and force field calculations pointing to an eta(2) coordination at Fe-6 of the FeMo-cofactor.