Báo cáo khoa học: Space, time and nitric oxide – neuronal nitric oxide synthase generates signal pulses pptx

Space, time and nitric oxide – neuronal nitric oxide synthase generates signal pulses John C Salerno1 and Dipak K Ghosh2 Biology Department, Kennesaw State University, GA, USA Department of Medicine, Hematology and Oncology, Duke University and Veterans Affairs Medical Center, Durham, NC, USA Keywords autoinhibition; diffusion; nitric oxide; nitric oxide synthase; pulse signaling Correspondence J C Salerno, Biology Department, Kennesaw State University, 1000 Chastain Road, Kennesaw, GA 30144, USA Fax: +1 770 423 6625 Tel: +1 770 423 6177 E-mail: jsalern3@kennesaw.edu (Received 23 June 2009, revised September 2009, accepted 15 September 2009) 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 °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 doi:10.1111/j.1742-4658.2009.07382.x Introduction Biological signaling takes place across spatial and temporal regimes spanning many orders of magnitude, and has applications in development, homeostasis, neuroscience and environmental studies Signaling and control theories have been extensively developed by electrical engineers and mathematicians [1] Their work underlies the design of many of the artifacts of our civilization, and is as germane to signaling and control in biology as it is to data transmission in a shielded cable or feedback control of temperature in a building The stability of positive and negative feedback loops in biological systems is governed by the same mathematical principles, which place stringent requirements on the gain and time response of components; this has become recognized in computational biology [2] but is not often considered in biochemistry Signal transduction is a developing area of explosive growth; in contrast, enzymology is, by any reasonable standard, a mature field Classic descriptions of activity (Michaelis–Menten [3], Cleland [4] and King-Altman [5]) rely on formalisms explicitly dependent on steady-state assumptions The incremental development of powerful analytical approaches has contributed greatly to the understanding enzymes in the steady-state, providing excellent descriptions of the ‘enzymes of mass conversion’ functioning in the interconversion of metabolites in biochemical pathways Signaling enzymes are well known and intensively studied [6,7] Signal generators differ from ‘metabolic enzymes’ in that, in addition to performing chemistry, they also transfer information Steady-state mass con- Abbreviations BH4, tetrahydrobiopterin; BTP, bis-tris propane; CaM, calmodulin; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; NHA, N-hydroxy arginine; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase; sGC, soluble guanylate cyclase FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS 6677 Space, time and nitric oxide J C Salerno and D K Ghosh version is the product of time and steady-state rate By contrast, information transfer in the unmodulated steady-state is zero because information transfer depends on bandwidth [8] Almost all work on signal transducing enzymes treats them as steady-state catalysts This is adequate only when the time regime is long and information transfer is slow Nitric oxide synthases (NOS) are signal generators in such diverse physiological processes as the control of vascular tone [9,10], signal transduction in the central nervous system [11–13] and the immune response [14–16] NOS (EC 1.14.13.39) generates NO from l-arginine, consuming mol of O2 and 1.5 mol of NADPH (three electrons) per mol of NO, and forming citrulline with N-hydroxy arginine (NHA) as an intermediate NO production by endothelial (eNOS) and neuronal (nNOS) isoforms is regulated by calmodulin (CaM) via the control of electron input to the catalytic site [17] NO activates soluble guanylate cyclase (sGC) and affects many other sites [18,19] Recent developments in NO signaling implicate important secondary targets in addition to sGC [19] The steady-state diffusion profiles of NO are notable for their shallow spatial gradients, enabling NO produced by eNOS to serve as a paracrine signal [20] NOS isoforms nonetheless target distinct receptors in nearby cells or even in the same cell [20] One of us recently suggested that the effective range of diffusion is limited by time-dependent signal production [21] Measurement of NO formation on a millisecond time scale is difficult However, the observation of heme intermediates provides an avenue for investigation of the time dependence of NO synthesis Results Global model and NO inhibition The product NO inhibits NOS [22] In iNOS and eNOS, inhibition is largely relieved by NO scavengers (e.g hemoglobin) [23]; relief by scavengers is less effective in nNOS Santolini et al [24,25] proposed a simplified ‘global model’ that accounts for the major features of NO inhibition The core of this model posits that quasi-geminate NO binds to ferriheme before escaping the active site Ferriheme NO (FeIII–NO) is unstable but, if an additional electron is delivered from FMN before NO escapes, stable ferrous NO forms As shown in Fig 1, FeIII–NO reduction and NO release partition the cycle at each turnover; relief of inhibition proceeds primarily by reaction with O2 [24,26] Strictly speaking, the rate constants in Fig connect rapid reaction segments, and not individual states These 6678 Fig Schematic of reaction cycle for NO synthesis [27] Rapid reaction segments are labeled by characteristic states All states are saturated with arginine or N-OH arginine (starred states), except the NO complexes, which are initially formed with citrulline bound Rate constants k1, k4 and k8 correspond to heme reduction; k2 and k5 correspond to oxygen binding; and k3 and k6 correspond to catalytic steps k7 and k9 correspond to the release of NO from ferric and ferrous enzyme, and k10 corresponds to the reaction of the ferrous NO complex with oxygen The long-lived FeII–NO complex is inhibitory reactions segments will be denoted here by the letters A–H, partly to emphasize this, but also because the reaction segments are characterized by different arginine derivatives and the states of the biopterin cofactors Ignoring this point leads to errors in chemistry The scheme shows the most significant states but omits the reaction intermediates and alternative states The cycle begins with the reduction of ferriheme (FeIII) (segment A) to ferroheme (FeII) (segment B), followed by O2 binding (leading to the initial FeII–O2 complex; segment C) The first oxygenase reaction forms NHA and FeIII (segment D); a second heme reduction (moving the system to segment E) triggers oxygen binding (forming the second FeII–O2 complex; segment F) and, subsequently, a second oxygenase reaction, forming NO and citrulline (initially present primarily as FeIII–NO; segment G) O2 binding and subsequent catalytic steps in both reactions are rapid compared to electron transfer Because of differences in the rate constants between isoforms, nNOS is more sensitive to quasi-geminate NO inhibition, and is approximately 80% inhibited when turning over in steady-state at 10 °C at high O2 tension ( 100 torr) [24] Reasonable projection of rate constants to 37 °C suggests that nNOS produces short ( 140 ms) pulses of NO [21]; simulations during the first second of catalysis are provided in Fig The differential equations describing this model are presented in Fig S1 Progressive formation of FeII–NO (segment H) causes a decay of NO production after the first turnover The sharp peak in NO production depends on the progressive formation of inhibitory FeII–NO Therefore, the hypothesis of pulsatile NO production at 37 °C can FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS J C Salerno and D K Ghosh Fig Simulations of NO production and population of states in the catalytic cycle at 37 °C Solid line, rate of NO formation (s)1); dashed line, fractional occupation of FeII–NO segment; black dash– dot line, total fractional occupation of FeII–O2 complexes; grey dash––dot line, fractional occupation of FeIII–NO complex Parameters: k1, k4 = 52 s)1; k8 = 28 s)1; k2, k5 = 520 s)1; k3, k6 = 100 s)1; k7 = 50 s)1; k9 = 0.01 s)1; k10 = 1.2 s)1 be critically tested by examining the formation of FeII–NO during the first few turnovers Spectra of intermediates As indicated in the preceding section, heme species with distinct spectra appear during catalysis These include high spin ferriheme, ferroheme and ferroheme O2 (each with either arginine or NHA); ferriheme NO; and ferroheme NO in the presence of citrulline (as formed) or arginine (after exchange) High spin ferriheme has a broad Soret band near 395 nm [27], shifting to 410 nm on reduction and 419 nm as the O2 adduct at low temperature [28] Ferroheme O2 formed after initiation of turnover at 10 °C has a Soret band at 427 nm [29] In arginine-saturated eNOS, the ‘hemeoxy II’ species has absorbance maxima near 432, 564 and 597 nm, but, with NHA bound, ferrous O2 peaks are significantly blue shifted to 428, 560 and 593 nm [30] In nNOS, ferric NO with arginine has a Soret maximum at 440 nm and has been reported to be similar to N-OH arginine; ferrous NO was reported with a Soret peak at 436 nm and a visible transition at 567 nm [31] As shown in Fig S2, we observed similar species We also prepared ferrous NO complex with saturating arginine and with mm citrulline The ternary ferroheme–arginine–NO complex is identical to the species described by Wang et al [31] with a Soret peak at 436 nm The spectrum of the ferrous–citrulline–NO complex is very similar (Fig S2, inset), allowing use of total FeII–NO in simulations Obvious changes in the trough of the Soret difference spectra are caused by Space, time and nitric oxide Fig Difference spectra of nNOS after s of turnover at 37 °C minus holoenzyme before activation of turnover in stopped flow (upper trace) and ferrous NO complex of nNOSox minus oxidized nNOSox, showing that the majority turnover species formed is the ferrous NO complex low spin ⁄ high spin thermal equilibrium in citrullinesaturated NOS Figure (upper trace) shows a difference spectrum obtained by subtracting the spectrum of nNOS, NADPH and arginine from the spectrum of nNOS, NADPH and arginine, s after initiation of turnover in air-saturated buffer ( 150 torr O2) The lower trace in Fig shows a difference spectrum of ferroheme NO of nNOS oxygenase domain minus the nNOS oxygenase with arginine The Soret and 567 nm bands are marked with arrows Clearly, the majority species formed during turnover at 37 °C is ferroheme NO, accounting for 74 ± 7% of total heme Figure shows the results of stopped flow experiments measuring the absorbance at 440 nm (lower trace) and 426 nm (upper trace) Wavelengths were chosen to maximize the contributions of the heme NO complexes (440 nm) and the ferroheme oxygen complex (426 nm) The 426 nm band has been fit to an exponential with a rate constant of 50 s)1; this adequately described the rise of the absorbance, which slightly falls off after 500 ms This is a reasonable measure of the time frame formation of the ferrous oxygen complex, which is limited by the rate of reduction of heme The absorbance at 440 nm is dominated by the ferric and ferrous NO complexes As shown in Fig 3, as the reaction progresses, the ferrous NO complex is dominant At short times, both complexes contribute, with the ferric NO complex forming slightly earlier in the first turnover The absorbance has been fit with two exponentials The majority contribution has a rate FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS 6679 Space, time and nitric oxide J C Salerno and D K Ghosh A Fig Stopped flow kinetics results at 37 °C showing traces at 426 nm (upper trace) and 440 nm (lower trace) after the initiation of turnover by mixing nNOS pre-reduced with NADPH and saturated with arginine with 200 lM calmodulin and mM CaCl2 The fits shown are the single exponential with 50 s)1 rate constant (426 nm) and two exponentials with 50 and s)1 rate constants in a : ratio Absorbance unit scales for 426 and 440 nm are shown on the right and left vertical axes, respectively constant of s)1, and contains contributions from both NO complexes A minority species accounting for the initial 25% of the absorbance change has a time constant of 50 s)1, most likely representing the tail of the ferrous oxygen complex band These rate constants are only descriptive because the reaction sequence is too complex to be modeled with a few exponentials (see simulations in Fig 5B, and in Fig 7B below) After the first 100 ms, a significant fraction of the enzyme is no longer in the initial turnover cycle In particular, the rise of the ferrous NO complex is not a single turnover event To obtain more information, spectra were recorded at ms intervals using an OLIS RMS rapid scan spectrometer The traces shown are from experiments initiated by mixing NADPH reduced nNOS with 200 lm CaM, which essentially eliminates complications from flavin spectra because both flavins are already reduced It is necessary to use high CaM concentrations because the on-rate for CaM is otherwise not fast enough to allow observation of the initial reactions It is possible that the initial reaction (heme reduction) is still somewhat affected by the CaM on-rate The rise of ferroheme NO is obvious in all data sets and is consistent with single wavelength kinetics at 440 nm Fitting of spectra at all positions to a three-component series yields initial and final ferric heme components and a ferroheme NO transient The rise of the transient corresponds to the falling edge of the NO pulse Additional components are indicated by residuals in the early spectra 6680 B Fig Kinetics of nNOS turnover of at 37 °C showing progressive FeII NO inhibition (A) Difference spectra of nNOS Soret region during turnover Baseline was averaged over the first four traces (0–3 ms traces), except the ms trace shows the unaveraged difference between and ms, which is reflected by the greater noise The major species are the 436–440 nm NO complexes, but an early transient can be seen near 425 nm (B) Time course of absorbance changes at 440 and 425 nm together with simulations based on the model in Fig Open circles, 440 nm absorbance; closed diamonds, 425 nm absorbance; dashed line, decay of fractional population of ferric heme; dotted line, time course of ferrous O2 complexes; light solid line, time course of ferrous NO complex; dash–dot line, rise of total heme NO complex; heavy solid line, combined absorbance of major states at 440 nm; medium solid line, combined absorbance of major states at 425 nm Simulation parameters: k1, k4 = 52 s)1; k8 = 28 s)1; k2, k5 = 1000 s)1; k3, k6 = 200 s)1; k7 = 50 s)1; k9 = 0.01 s)1; k10 = s)1 To improve signal to noise, spectra were averaged over intervals of 10 ms after the first few traces As shown in Fig 5A, after the initiation of catalysis, a Soret component at 427 nm rapidly forms, reaching a maximum at  20 ms; this is followed by the rise of longer wavelength components, which reach half maximal intensity after  50 ms Long wavelength components increase slowly until  500 ms, reaching a plateau that extends until reagents are depleted It is FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS J C Salerno and D K Ghosh likely that, during the initial few 100 ms, ferroheme NO is formed in the presence of citrulline but, at times longer than s, the dominant ferrous NO complex is the arginine bound state Figure 5B shows a plot of absorbance at three wavelengths with simulations based on the model shown in Fig Simulations are derived from a Runge–Kutta numerical solution of the differential equations describing the model as previously described [24] Ferriheme declines from an initial value of 100% of heme, reaching a steady-state of approximately 7% The simulation only approximates exponential decay, primarily as a result of the contribution of ferriheme N-OH arginine in segment D The first transient shown is the ferrous O2 complex, which has two components (segments C and F) corresponding to the two oxygenase reactions Hence, the first component is the arginine ferrous O2 complex and the second the N-OH arginine ferrous O2 complex The second transient is ferriheme NO, which has only a single component (segment G); ferroheme NO increases monotonically, approximating an exponential after a 20 ms lag The difference absorbance at 440 nm (open circles) can be approximated by the sum of contributions from segments G and H (ferrous and ferric NO complexes) at times longer than 20 ms; at short times, there is a small component from ferroheme O2 The simulation shown includes equal contributions from these species Between 422 and 430 nm, absorbance changes are dominated by segments C and F (ferrous oxy complexes) with some apparent contribution from segment G The ferroheme contribution (not shown) is small, peaking before 10 ms The parameters employed in Fig not represent a unique fit However, the heme reduction rate is consistent with projection from direct measurements at lower temperatures, and the data presented require an initial heme reduction rate in the range 45–60 s)1 to account for ferroheme O2 formation (but see also the three-electron model in the Discussion) The rates of O2 binding and catalysis are similarly constrained (primarily at the low end), and the maximum 37 °C steady-state turnover rate (2–3 s)1) provides an additional cross check Figure shows the temperature dependence of the rate of heme reduction in nNOS holoenzyme using several experimental approaches These include stopped flow measurements of heme-CO derivatives, single wavelength and spectral measurements made during early turnover, and flash experiments initiating electron transfer by CO dissociation [32] Heme reduction is reasonably well described in this regime by a single activation energy of  80 kJỈmol)1 NADPH Space, time and nitric oxide Fig Temperature dependence of electron transfer rate in nNOS holoenzyme Rates of heme reduction at 10 °C are estimated from CO binding stopped flow experiments [17] and at 15, 25 and 37 °C from turnover stopped flow experiments (present study) The rate at 22 °C is taken from a flash dissociation initiated experiment [32] and CaM initiated rates fall on the same plot, indicating that any effects of CaM binding rates on the initiation of electron transfer are secondary Although the quality of the data does not yet allow deeper analysis of intermediates formed in the first 50 ms, it is clear that steady-state turnover at 37 °C is inhibited by the formation of a majority ferrous NO complex, leading to production of a pulse of NO during the initial few turnovers in 100–200 ms It is equally clear that the heme reduction rate is at least  50 s)1, and is not much faster than 70 s)1 Figure illustrates the results of experiments conducted at 22 °C to investigate the ability of nNOS to produce multiple pulses in sequence Figure 7A shows a sequence in which nNOS turning over in steady-state is repeatedly stopped and started by the sequential addition of the chelator EDTA and calcium The initial sequence shows the steady-state consumption of NADPH; pulses of higher activity are elicited by stopping the reaction with EDTA and restarting with Ca2+ after an interval of 1–10 s Neither EDTA nor Ca2+ alone produces a pulse Simultaneous addition of EDTA and Ca2+ (with a slight excess of EDTA) produces a small pulse expected for a mixing time of 0.5–1.0 s Figure 7B shows a similar experiment expanded to show the details of the pulses The top trace shows the effect of adding Ca2+ to nNOS turning over in steadystate; no pulse is produced because quasi-geminate NO FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS 6681 Space, time and nitric oxide J C Salerno and D K Ghosh and is consistent with the slightly greater inhibition observed at 22 °C than at 10 °C Strong pulses can be elicited even after the steady-state activity slows down (not shown), presumably because oxygen consumption slows down the steady-state activity before the initial rate decreases A Discussion Three-electron models B Fig Sequential pulse formation during NO synthesis by nNOS at 22 °C, monitored by measuring NADPH consumption at 340 nm Pulses were generated by stopping steady-state reaction with 60 lM EDTA and, after a delay, restarting turnover by addition of 60 lM CaCl2 Solutions were mixed with a 500 lL gas tight Hamilton syringe Initial conditions were: lM nNOS in 50 mM Mops (pH 7.5), 50 mM KCl, 10% glycerol, 200 lM arginine, lM calmodulin (A) Segments from upper left represent steady-state turnover, pulse generated by starting and stopping with a s interval, pulse after a s interval, pulse after simultaneous injection of EDTA and Ca+2, pulse after s interval, and pulse after s interval (B) Segments selected from a separate experiment showing pulse regions in detail Open circles, lack of a pulse when extra Ca2+ is added without stopping the reaction; filled diamonds, pulse after s interval; X characters, s interval; filled triangles, s intervals; open squares, s interval Dashed lines represent the initial (maximum) rate of the fully developed pulse, steady-state rate at high ( 120 torr) pO2, and the initial rate of pulse after a s interval is continuously generated in steady-state The other traces show pulses that increase with the interval between EDTA and Ca2+ addition The lines represent the steady-state rate, the initial pulse rate after full recovery, and the initial pulse rate after s recovery The maximum pulse rate is approximately eight-fold greater than the steady-state rate, and approximately half the maximum pulse can be elicited after 3–4 s The recovery of pulse intensity has a rate constant of 0.25 ± 0.05 s)1 This compares reasonably well to the s)1 rate constant used for the reaction of oxygen with the ferrous NO complex in 37 °C simulations, 6682 Simulations based directly on the original Santolini model [24] account for the observations made during turnover at 37 °C at the present level of detail, demonstrating pulsed catalysis under these conditions This confirms the central premise of the model: the progressive inhibition by formation of the ferrous NO complex from quasi-geminate NO The original model does not account for all features of the catalytic cycle In particular, only two steps in the productive loop of the model correspond to electron transfer from FMN to heme The reactions producing NO from arginine require three electrons The first oxygenase reaction, producing N-OH arginine from arginine, requires two electrons The first electron is explicitly accounted for in the initial step, in which the enzyme is primed for O2 binding by heme reduction The second electron is supplied by tetrahydrobiopterin (BH4) [33,34]; the reductive reaction is subsumed into a catalytic step with rate constant k3 BH4 and heme are closely associated within the oxygenase domain [35], and thermodynamically favorable electron transfer between them should be rapid in comparison to shuttle delivery of electrons via FMN [36] The second oxygenase reaction requires only one electron to prime heme for O2 binding The initial oxygenase reaction leaves biopterin as a one electron oxidized radical, which must be subsequently reduced to BH4 for turnover to continue The electron is supplied from NADPH via FMN BH4 regeneration is not included in the original two-electron model Several three-electron models that retain inhibitory feedback differ in the position in which BH4 is regenerated In Fig 8A, regeneration occurs immediately after the first oxygenase reaction Heme reduction is followed by rapid biopterin reduction, so ferriheme predominates in D and D1 The rate constant essentially describes heme reduction by FMN This is followed immediately by ferriheme reduction, priming heme for O2 binding to start the second oxygenase reaction Differential equations describing the model are given in Fig S3, in addition to two other examples (Figs S4 and S5) In the first, the electron for BH4 regeneration is supplied FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS J C Salerno and D K Ghosh A B Space, time and nitric oxide simulation of Fig 5B Using this model, the best fits are obtained with somewhat faster rates for the second and third reductive steps, and a slightly slower rate of catalysis for the first oxygenation One possibility is that the initial electron transfer reaction is slightly affected by a 10 ms delay caused by the rate of binding of calmodulin We not claim to be able to estimate all the parameters to high accuracy from these simulations because the simulations are insensitive to some rates as long as they are fast, and because parameters (other than the rates of heme reduction) can often be be adjusted by a factor of two to three It is not yet feasible to discriminate between three-electron models based on fitting Although two-electron models account for kinetics, they should be replaced by three-electron models that explicitly account for stoichiometry Physiological implications Fig Three-electron model for NO catalytic cycle (A) Rapid reaction segments are labeled A–H instead of labeling by characteristic states All states are assumed to be saturated with arginine or N-OH arginine (starred states) except the NO complexes, which are initially formed with citrulline bound Rate constants k1, k4, k¢4 and k8 correspond to heme reduction; k2 and k5 correspond to oxygen binding; and k3 and k6 correspond to catalytic steps k7 and k9 correspond to release of NO from ferric and ferrous enzyme, and k10 corresponds to reaction of the ferrous NO complex with oxygen Segment D is characterized by ferric heme, biopterin radical and N-OH arginine, whereas, in segment D1, the characteristic state is ferric heme in the presence of BH4 and N-OH arginine because the first electron entering heme after N-OH arginine formation regenerates BH4 The long-lived FeII–NO complex (segment H) is inhibitory (B) Simulations of absorbance kinetic data using the three-electron model of Fig 8A Open circles, 436 nm absorbance data; open triangles, 425 nm absorbance data; closed diamonds, 425 nm absorbance data; dashed line, decay of fractional population of ferric heme; dotted line, time course of ferrous O2 complexes; light solid line, time course of ferrous NO complex; dash– dot line, rise of total heme NO complex; heavy solid line, combined absorbance of major states at 440 nm; medium solid line, combined absorbance of major states at 425 nm Parameters: k1 = 45 s)1; k4, k¢4 = 70 s)1, k8 = 70 s)1; k2, k5 = 800 s)1; k3 = 70 s)1, k6 = 80 s)1; k7 = 120 s)1; k9 = 0.02 s)1; k10 = 0.5 s)1 during catalysis via an unspecified intermediate, producing an early appearance of the second ferroheme oxygen complex In the second model, BH4 regeneration can occur either before or after oxygen complex formation, producing a pathway branched at both the regenerative step and the inhibitory loop Figure 8B shows a simulation of experimental data from 37 °C kinetics experiments with the three-electron model of Fig 8A A reasonable fit was obtained by slight adjustment of kinetics parameters from the The experimental results presented here demonstrate that nNOS is capable of pulsed production of NO at 37 °C, and provide an initial characterization of the state of the enzyme during pulse formation and progressive inhibition Pulsatile behavior of nNOS occurs even in air-saturated buffer at an O2 tension of  150 torr Physiological O2 tension varies greatly between tissues and, in some tissues, varies greatly between physiological states Tissue pO2 varies from approximately 100 torr (in the lung) to 20 torr [37], and falls during stress or exercise As pO2 falls, the removal of the ferrous NO complex slows; ferrous NO formation from the ferric NO branch point is independent of O2 tension The inhibited state thus becomes increasingly prevalent Simulations using the models presented here suggest that, in steady-state at 37 °C and 40 torr, nNOS is inhibited by more than 90%, assuming that the removal of ferroheme NO is first order in O2 Further details will be made available in subsequent studies Pulsatile behavior of nNOS is therefore likely to be even more pronounced at physiological pO2 The effective Km for O2 is much lower for pulsed NO production, and dominated by oxygen binding to ferrous heme, than for steady-state NO production, in which the relief of inhibition is critical As a result, nNOS steady-state activity is a significant fraction (20–30%) of the uninhibited rate at the highest physiological oxygen tensions but is a much smaller fraction at lower O2 tensions, whereas pulsed NO production is almost maximal (for example, see [25]) As pointed out by several groups, the apparent Km for nNOS is anomalously high for a heme oxygenase; Santolini et al [24] FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS 6683 Space, time and nitric oxide J C Salerno and D K Ghosh have attributed this to the relief of inhibition The presteady-state rate of NO synthesis is essentially O2-independent until hypoxic levels of oxygen are attained As noted previously [21], the pulsatile behavior of nNOS produces very sharp spatial gradients of NO that limit the range of NO signaling As an example of the spatial and temporal character of NO diffusion from a pulse, Fig S6 provides a simulation of diffusion during and following a pulse from a generating volume of eukaryotic dimensions (i.e a sphere 20 lm in diameter with internal layers lm thick) The pulse function used includes a 15 ms delay, a rapid (5 ms) rise and a 140 ms exponential decay The pulse is truncated at 210 ms to produce a small marker discontinuity Diffusion alone quickly reduces the NO concentration in volume elements of cellular dimensions or smaller, such that pulses in NO production always lead to pulses in NO concentration in systems of that magnitude The concentration pulse produced here is approximately twice the duration of NO synthesis, and is limited to approximately 50 lm with respect to the effective range Sharper pulses and smaller generating areas produce sharper pulses in space and time A small nNOS array in a postsynaptic region would produce very short ranged pulsed signals We hope to explore this topic in subsequent studies Other isoforms Quasi-geminate NO inhibits iNOS and eNOS less than nNOS because of differences in the rate constants for the reduction of steady-state ferroheme NO [27] At 10 °C and 100 torr, steady-state activity in these isoforms is only marginally inhibited, such that NO formed in pre-steady-state catalysis is negligible compared to NO formed in steady-state However, at low pO2, inhibition by quasi-geminate NO can be significant In constitutively active iNOS, this primarily reduces steady-state activity at low pO2 [24] Because eNOS, similar to nNOS, is activated by CaM and phosphorylation, it presents the possibility of significant steady-state and pre-steady-state phases of catalysis Simulations of NO synthesis by eNOS using the models presented here indicate that, as pO2 falls, progressive inhibition of steady-state catalysis makes the pre-steadystate more significant in comparison Pre-steady-state production of NO by eNOS at low pO2 is, however, quite different than the pre-steady-state production of NO by nNOS Because heme reduction in eNOS is more than one order of magnitude slower than heme reduction in nNOS, eNOS is incapable of generating sharp pulses unless an additional factor (e.g phosphorylation) greatly increases electron transfer In 6684 particular, phosphorylation of sites such as S615 and S633, associated with the autoinhibitory element, and S1177, associated with the C terminal tail regulatory site, increases the electron transfer rate and should preferentially promote presteady-state NO synthesis [38] At low O2 (e.g 20 torr) eNOS simulations suggest that it generates NO for several seconds and is progressively inhibited; inactivation kinetics and the degree of steady-state inhibition depend on the model and the O2 tension, but it is likely that eNOS is often inhibited by 75–80% in the steady-state Because inactivation is slow, the persistence of pre-steady-state rates of NO synthesis to low O2 tensions produces extended NO production rather than sharp  100 ms pulses Effectively, the pre-steady-state Km for O2 is lower than the steady-state Km Because pre-steady-state NO synthesis is extended, diffusion of the NO signal is much less restricted Instead, the negative feedback loop limits the concentration of NO produced by active eNOS The higher activity of eNOS in pre-steady-state serves to rapidly establish a level of NO that is capable of activating sGC The pre-steady-state synthesis of NO by eNOS, and its effect on vascular diffusion patterns, will be examined in future studies Pulsed signal generators It should be clear that production of signal pulses is not dependent on the details of catalytic cycle models, but reflects instead the progressive inhibition of a signal generator after activation In the previous model [24] and in the closely-related three-electron models introduced here, feedback inhibition is produced by quasi-geminate NO, a confined reaction product The key element is the production of an additional inhibited state, the ferrous NO complex, which is not the parent of free NO in the productive cycle The progressive accumulation of a stable enzyme– product complex is one theme that might recur in other signal generators However, this need not involve product inhibition A more general view of pulse generation is presented in Fig The signal generator is initially in a resting state ER; with activation in response to a stimulus produces state EA EA generates a signal but decays to state EI As the inhibited state EI accumulates, the rate of signal production falls, limiting the extent of the pulse The signal generator is turned off by removal of the stimulus or the introduction of an antagonist, and EI is converted to EIR EIR in turn decays to the resting state ER In NOS, EA is produced by CaM binding in response to calcium, and corresponds to the enzyme in FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS J C Salerno and D K Ghosh the productive catalytic cycle EI corresponds to the inhibited enzyme tied up as ferrous NO complex Removal of calcium or phosphorylation stops electron transfer (state EIR) and reaction with oxygen gradually restores the original resting state during a latency period Calcium removal and enzyme inactivation are rapid events, occurring on a millisecond time scale; as shown in the present study, the decay of the enzyme to the resting state occurs on a s time scale The pulses of NO in simulations presented in the previous study [21] are strikingly similar in shape to action potentials and other transport-like transients, although they are much slower The mechanism responsible for the generation of these potentials provides an interesting parallel to pulsed signal generation in NOS Sodium and potassium channels open in response to partial membrane depolarization, and ion currents rapidly depolarize the membrane This corresponds to activation The inhibitory event is conformational, corresponding to binding of an autoinhibitory element by activated channels This closes the channels within milliseconds and allows membrane repolarization, deactivating the channels The autoinhibitory element is released in response to deactivation, regenerating the resting inactive configuration [39] This is a rapid process (millisecond time scale) The refractory period limits the frequency of pulses and the rate of information transfer Limitations imposed by rate constants of the molecular elements necessitate massive parallelism in sensory and motor pathways NO is a retrograde signal in the central nervous system [40] Because the refractory period of NO pulses is much longer than the refractory period of neurons, NO pulses cannot form spike trains on the time scale Space, time and nitric oxide of action potential spike trains An NO ‘spike’ lasts 50–100 times as long as an action potential It is more productive to consider it as a response to the initiation of synaptic activity rather than a translated action potential Keller et al [41] recently simulated calmodulin release and activation in synapses, using calcium-sensitive dye recordings on a millisecond time scale Applying their results to nNOS activation, it is clear that calcium spikes and high local CaM concentrations are more than sufficient to activate an array of nNOS molecules bound at the synapse through their PDZ domains The time frame of the NO pulse is appropriate for a retrograde signal (e.g one that functions as a mediator in synaptic plasticity) Because signaling is inherently time-dependent, pulse formation on different time scales may be common to many signal generators The appropriate time domain is determined by many factors, including the information transfer rate, the time frame of feedback loops that depend on the signals, and the distance over which a diffusible molecular signal acts For slowlymodulated signals, low frequency pulses can be produced using feedback from downstream processes Short pulses are likely to require internal feedback, as in the nNOS and potassium channel examples Materials and methods Expression and purification of nNOS DNA encoding rat nNOS holoenzyme, a gift from Dr S Snyder (Johns Hopkins University, Baltimore, MD, USA), was cloned in pCWori+ [42,43] Rat nNOS was expressed in Escherichia coli strain BL21DE, and purified using ammonium sulfate precipitation and gel filtration and 2¢,5¢-ADP Sepharose chromatography [43,44] Activity was measured by oxyhemoglobin assay, and was 500–700 nmolỈmin)1Ỉmg)1 protein [43–45] Purified nNOS contained 0.8–1.0 hem mol)1 (CO difference spectra extinction coefficient of 74 mm)1Ỉcm)1) and FMN and FAD contents after extraction from nNOS were at least 90% of heme Rat nNOSoxy was expressed and purified as reported previously [44,45] Spectroscopy Fig Scheme for internally regulated pulse signal generation ER denotes a resting state; activation produces the active state EA EA generates a signal but decays to the inhibited state EI EI accumulation leads to the decay of the pulse After inactivation (e.g removal of stimulus or introduction of an antagonist), EI is converted to the transient state EIR EIR decays to the resting state ER The binding of l-arginine, H4B and citruline were monitored by UV-visible spectral perturbation Absorbance spectra of purified NOS oxygenase and holo proteins were obtained using a Hitachi U2010 Spectrometer (Hitachi, Tokyo, Japan) and data collection software (UV solutions, Wellesley Hills, MA, USA) Ferric and ferrous nitrosyl complexes were produced at 25 °C in 40 mm bis-tris FEBS Journal 276 (2009) 6677–6688 ª 2009 The Authors Journal compilation ª 2009 FEBS 6685 Space, time and nitric oxide J C Salerno and D K Ghosh propane (BTP) buffer, pH 7.5, 150 mm NaCl, 10 lm H4-biopterin in the presence of either mm l-arginine or citruline, or both The final NOS concentration was 3–4 lm, and NO was generated by the decay of PROLINONOate (0.2 mm; Alexis Biochemicals, San Diego, CA, USA), an NO donor Spectra were recorded at 3600 nmỈmin)1; after collecting oxidized spectra, dithionite was added to obtain ferrous nitrosyl spectra Difference spectra were obtained by digital subtraction Pulse trains were generated at 22 °C using a 500 lL Hamilton syringe for rapid mixing; the solution was quickly withdrawn from the cuvette and reinjected along with an aliquot of the reagent to be added The mixing time was approximately 0.5 s, and the minimum interval between additions was approximately s Kinetics Kinetics experiments were conducted using an Applied Photophysics SX stopped flow unit (single wavelength) (Applied Photophysics, Leatherhead, UK) or an OLIS RMS-1 rapid scan spectrophotometer (OLIS Instruments, Bogart, GA, USA) equipped with a stopped flow device Reactions were initiated by mixing 3–6 lm solutions of nNOS, mm arginine and 200 lm NADPH in air-saturated BTP at pH 7.5 with 120 lm calmodulin and mm CaCl2, or by mixing lm solutions of nNOS, mm arginine, 12 lm calmodulin and 100 lm CaCl2 in air-saturated BTP at pH 7.5 with mm NADPH Data collection and preliminary analysis of spectral stopped flow data was performed using olis proprietary software 10 11 Simulations Simulations of kinetics data were carried out as described previously [24] The systems of first-order differential equations describing the kinetic behavior of the models were solved by numerical integration using fourth-order Runge– Kutta methods Simulations were checked by systematically setting all rate constants to zero except one, and by comparison with Euler’s method programs 12 13 14 Acknowledgements This work was supported by NIH GM083317-01 (J.S.) and Axxora LLC (D.G.) 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