Dynamics of NO interacting with soluble guanylate cyclase from 1 ps to 0.1 s and induced structural transitions

1471-2210-11-S1-P77 1471-2210 Poster presentation Dynamics of NO interacting with soluble guanylate cyclase from 1 ps to 0.1 s and induced structural transitions YooByung-Kuk LamarreIsabelle MartinJean-Louis RappaportFabrice NegrerieMichelmichel.negrerie@polytechnique.fr

Laboratoire d'Optique et Biosciences, INSERM, Ecole Polytechnique, Palaiseau, France

Institut de Biologie Physico-Chimie, CNRS, Paris, France

BMC Pharmacology 5th International Conference on cGMP: Generators, Effectors and Therapeutic ImplicationsJohn Burnett Jr, Franz Hofmann, Harald HHW Schmidt and Johannes-Peter StaschMeeting abstracts - A single PDF containing all abstracts in this supplement is available here.5th International Conference on cGMP: Generators, Effectors and Therapeutic ImplicationsHalle, Germany

24-26 June 2011

http://www.cyclicgmp.net/1471-2210 2011 11 Suppl 1 P77 http://www.biomedcentral.com/1471-2210/11/S1/P77 10.1186/1471-2210-11-S1-P77 182011 2011Yoo et al; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Results

We investigated the interaction between purified soluble guanylate cyclase (sGC) from beef lung and NO by time-resolved spectroscopy in a time-range which encompasses eleven orders of magnitude, from 1 ps 1 to 0.1 s 2 . After its dissociation from the heme, NO either recombines geminately to the 4-coordinate heme within τ_G1 = 7 ps 1 (96 ± 1 % of the population) or exits the heme pocket (4 ± 1 %), allowing the proximal histidine to rebind within 62 ± 10 ps. Then, NO is distributed in two approximately equal populations (~2 %). One geminately rebinds to the 5-coordinate heme (τ_G2 = 6.5 ns) while the other migrates into the solution. NO can rebind from the solution (bimolecular rebinding, τ_B = 0.25 ms with [NO] = 20 µM), forming a 6-coordinate heme with a rate constant of 2 x 10⁸ M^-1 s^-1 (in vitro purified protein) very close to that measured in platelets (3 × 10⁸ M^-1s^-1) 3 .

The cleavage of Fe-His bond and subsequent formation of 5-coordinate NO-heme occurs with different time constants for NO which geminately rebinds (τ_5C1 = 0.66 µs) and for NO which binds from the solution (τ_5C2 = 43 ms). Thus, because the same structural event occurs with rates separated by more than 4 orders of magnitude, we must infer that sGC is not in the same structural state in both cases, with a different strain exerted on the Fe-His bond. This allosteric transition between both states occurs in the time range 0.66 µs < τ_R < 250 µs in sGC after His rebinding and NO release.

Conclusion

Since the discovery that NO binds to the proximal heme side in cytochrome c' 4 (AXCP), several models of sGC activation were proposed which include the binding of NO to the proximal heme side despite the lack of observation for such an activation step in sGC. After the fast histidine rebinding in the picosecond range, we have observed only four phases in the nano to millisecond time range (assigned as indicated in Table 1). Thus, analysis of the entire NO dynamics from 1 ps to 0.1 s did not detect NO binding to the proximal side of sGC heme despite the fact that NO shows the same geminate rebinding to the 4-coordinate heme in sGC and AXCP 5 . Our data can be described with a one-site model, without phases assigned to dinitrosyl formation or to NO proximal binding.

Table 1

Rates of the transitions observed in kinetics.

Transition

Time constants

Transition rates

Bimolecular NO binding to 5c-His

τ_B = 0.25 ms; [NO]= 20 µM

k_B = 2 x 10⁸ M^-1s^-1

Conversion 6c-NO → 5c-NO

τ_5C2 = 43 ms

k_5C₂ = 23 s^-1

Geminate NO rebinding to 5c-His

τ_G2 = 6.5 ns

k_G2 = 0.15 x 10⁹ s^-1

Conversion 6c*-NO → 5c*-NO

τ_5C1 = 0.66 µs

k_5C1 = 1.5 x 10⁶ s^-1

His rebinding to 4c-heme

τ_His = 62 ps

k_His = 1.4 x 10¹⁰ s^-1

Geminate NO rebinding to 4c-heme

τ_G1 = 7 ps

k_G1 = 0.13 x 10¹² s^-1

Structural relaxation sGC* → sGC

0.66 µs < τ_R < 250 µs

4 x 10³ s^-1 <k_R <1.5 x 10⁶ s^-1

Figure 1

Model describing the species and transitions involved in the time-resolved experiments. a: 5-c-His sGC in the resting state. b: 6-C sGC. c: 5-c-NO sGC, in the activate state. d: 4-c sGC after NO dissociation. e: 5-c-His sGC still in the activated conformation, immediately after His105 rebinding. f: 6-c sGC in the activated state, immediately after NO geminate rebinding. The value adjacent to the letter label is the wavelength of the species. The star at the right bracket in c, d, e denotes the activated state of sGC. The starting species of the kinetic measurements is c. The time constants indicated are those measured and the corresponding rates are given in Table 1. In species d, NO is located within the heme pocket whereas in species e, NO is located in another docking site in the protein.

Control of nitric oxide dynamics by guanylate in its activated stateNegrerieMBouzhirLMartinJLLieblUJ Biol Chem2001276468154682110.1074/jbc.M10222420011590135A new high-sensitivity 10-ns time-resolution spectrophotometric technique adapted to in vivo analysis of the photosynthetic apparatusBealBRappaportFJoliotPRe Sc Instrum19997020220710.1063/1.1149566Nitric oxide activation of guanylyl cyclase in cells revisitedRoyBGarthwaiteJPro. Natl Acad Sci USA2006103121851219010.1073/pnas.0602544103Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclaseLawsonDMStevensonCEMAndrewCREadyREMBO J2000195661567110.1093/emboj/19.21.566130580611060017Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c'KruglikSGLambryJCCianettiSMartinJLEadyRRAndrewCRegrerieMJ Biol Chem20072825053506217158883