; Dynafit script used for fitting and simulations of pre-steady state reduction of cytochrome c1 and b

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1 SUPPLEMENTAL DATA ; Dynafit script used for fitting and simulations of pre-steady state reduction of cytochrome c1 and b [task] task = simulate ; or fit when optimizing parameters marked with "?" data = progress ; E = oxidized bc1 complex ; EFeSb = Rieske protein and one b heme reduced ; Ecb = cyt c1 and one b heme reduced ; EFeSbb = Rieske protein and two b hemes reduced ; Ecbb = cyt c1 and two b hemes reduced ; QH2 = decylubiquinol ; Q = decylubiquinone ; units in s-1 and microm [mechanism] ; first turnover E + QH2 <===> E.QH2 : kaq kdq E.QH2 <===> EFeSb.Q : k1 k_1 EFeSb.Q <===> Ecb.Q : kfes k_fes EFeSb.Q <===> EFeSb + Q : kdq2 kaq EFeSb <===> Ecb : kfes k_fes Ecb.Q <===> Ecb + Q : kdq2 kaq ; second turnover Ecb + QH2 <===> Ecb.QH2 : kaq kdq Ecb.QH2 <===> EFeScbb.Q : k1 k_1' EFeScbb.Q <===> EFeScbb + Q : kdq2 kaq EFeSb + QH2 <===> EFeSb.QH2 : kaq kdq EFeSb.QH2 <===> Ecb.QH2 : kfes k_fes

2 [constants] kaq = 5000 kdq = 50000? ; or 85000? for Y185F kdq2 = 20000? ; or 40000? For Y185F k1 = 50? ; or 10? for Y185F k_1 = 2.8 ; or 4.5 for Y185F k_1' = 6.25 ; 2.8 assuming crossover, for Y185F: 10 assuming no crossover, 4.5 with crossover kfes = k_fes = [responses] ; extinction coefficients x2, due to 2 cm pathlength ; when measuring cyt c1 (ext. coeff = 17.5 mm-1 cm-1): Ecb = Ecb.QH2 = Ecb.Q = EFeScbb = EFeScbb.Q = ; when measuring cyt b (ext. coeff = 35 mm-1 cm-1 for bh, 15 mm-1 cm-1 for bl): EFeSb = 0.07 EFeSb.QH2 = 0.07 EFeSb.Q = 0.07 Ecb = 0.07 Ecb.QH2 = 0.07 Ecb.Q = 0.07 EFeScbb = 0.14 ; or 0.1 assuming no crossover EFeScbb.Q = 0.14 ; or 0.1 assuming no crossover [progress] directory extension mesh./anti/data txt from 0 to 2 step 0.05 ; or from 0 to 6 for Y185F

3 file antcytc05 concentration QH2 = 0.75, E = 0.75 ; or 1.5 file antcytc1 concentration QH2 = 1.5, E = 0.75 ; or 1.5 file antcytc2 concentration QH2 = 3, E = 0.75 ; or 1.5 file antcytc4 concentration QH2 = 6, E = 0.75 ; or 1.5 [output] directory./anti/output [end]

4 ; Dynafit script used for fitting and simulations of pre-steady state reduction of exogenous cytochrome c and of cytochrome b [task] task = simulate ; or fit when optimizing parameters marked with "?" data = progress ; E = oxidized bc1 complex ; EFeSb = Rieske protein and one b heme reduced ; Ecb = cyt c1 and one b heme reduced ; EFeSbb = Rieske protein and two heme b reduced ; Ecbb = cyt c1 and two heme b reduced ; QH2 = decylubiquinol ; Q = decylubiquinone ; cox = oxidized cytochrome c ; cred = reduced cytochrome c ; SQ = semiquinone ; units in s-1 and microm [mechanism] ; first turnover E + QH2 <===> E.QH2 : kaq kdq E.QH2 <===> EFeSb.Q : k1 k_1 EFeSb.Q <===> EFeSb + Q : kdq2 kaq EFeSb <===> Ecb : kfes k_fes Ecb + cox <===> Ecb.cox : kac kdc Ecb.cox <===> Eb.cred : kc k_c Eb.cred <===> Eb + cred : kdc kac ; second turnover Eb + QH2 <===> Eb.QH2 : kaq kdq Eb.QH2 <===> EFeSbb.Q : k1 k_1' EFeSbb.Q <===> EFeSbb + Q : kdq2 kaq EFeSbb <===> Ecbb : kfes k_fes Ecbb + cox <===> Ecbb.cox : kac kdc Ecbb.cox <===> Ebb.cred : kc k_c Ebb.cred <===> Ebb + cred : kdc kac

5 ; antimycin insensitive rate Ebb + QH2 <===> Ebb.QH2 : kaq kdq Ebb.QH2 <===> EFeSbb.SQ : k1 k_2 EFeSbb.SQ <===> EFeSbb + SQ : kdq3 kaq SQ ----> Q : k3 [constants] kaq = 5000 kdq = 65000? ; or ? for Y185F kdq2 = 35000? ; or 5000? for Y185F kdq3 = 12000? ;1500? for Y185F kac = kdc = k1 = 50? ; or 10? for Y185F k_1 = 2.8 ; or 4.5 for Y185F k_1' = ; 2.8 assuming crossover, for Y185F: 10 assuming no crossover, 4.5 with crossover kfes = k_fes = kc = k_c = k_2 = ; or for Y185F k3 = ; or for Y185F [responses] ; extinction coefficients x2, due to 2 cm pathlength ; when measuring cyt c (ext. coeff= 21.5 mm-1 cm-1): Eb.cred = Ebb.cred = cred = 0.043

6 ; when measuring cyt b (ext. coeff= 35 mm-1 cm-1 for bh, 15 mm-1 cm-1 for bl): Eb = 0.07 Eb.QH2 = 0.07 Eb.cred = 0.07 EFeSb = 0.07 EFeSb.Q = 0.07 Ecb = 0.07 Ecb.cox = 0.07 Ecbb = 0.14 ; or 0.1 assuming no crossover Ecbb.cox = 0.14 ; or 0.1 assuming no crossover Ebb.cred = 0.14 ; or 0.1 assuming no crossover EFeSbb = 0.14 ; or 0.1 assuming no crossover EFeSbb.Q = 0.14 ; or 0.1 assuming no crossover Ebb = 0.14 ; or 0.1 assuming no crossover Ebb.QH2 = 0.14 ; or 0.1 assuming no crossover EFeSbb.SQ = 0.14 ; or 0.1 assuming no crossover [progress] directory extension mesh./anti/data txt from 0 to 2 step 0.05 ; or from 0 to 6 for Y185F file cantcytc05 concentration cox = 9, QH2 = 0.75, E = 0.75 ; or 1.5 file cantcytc1 concentration cox = 9, QH2 = 1.5, E = 0.75 ; or 1.5 file cantcytc2 concentration cox = 9, QH2 = 3, E = 0.75 ; or 1.5 file cantcytc4 concentration cox = 9, QH2 = 6, E = 0.75 ; or 1.5 [output] directory./anti/output [end]

7 ; Dynafit script used for fitting of inhibition of steady-state activity by antimycin assuming activation of one monomer upon binding of one antimycin, and electron crossover between cyt b subunits [task] task = fit data = velocities model = activation of monomer and crossover ; E = bc1 dimer ; S = substrate (ubiquinol) ; I = inhibitor (antimycin) ; P = product (ubiquinone) ;? = fitted variable ; all concentrations were multiplied by 20 to normalize E to 1 (actual [E] of 0.05 microm) [mechanism] ; substrate and inhibitor equilibration in the dimer: E + S <===> ES ES + S <===> SES E + S <===> SE SE + S <===> SES E + I <===> EI E + I <===> IE EI + I <===> IEI IE + I <===> IEI EI + S <===> ESI EI + S <===> SEI IE + S <===> IES IE + S <===> ISE ES + I <===> ESI ES + I <===> IES SE + I <===> SEI SE + I <===> ISE

8 IEI + S <===> IESI IEI + S <===> ISEI SES + I <===> SESI SES + I <===> ISES ESI + S <===> SESI IES + S <===> ISES SEI + S <===> SESI ISE + S <===> ISES ESI + I <===> IESI IES + I <===> IESI SEI + I <===> ISEI ISE + I <===> ISEI SESI + I <===> ISESI ISES + I <===> ISESI ISEI + S <===> ISESI IESI + S <===> ISESI ; catalytic steps: ES ---> E + P : kcat ; only one monomer is active SES ---> SE + P : kcat SEI ---> EI + P : kcat ; inactive monomer becomes active IES ---> IE + P : kcat ESI ---> EI + P : kcat ; crossover allows activity of antimycin-bound monomer ISE ---> IE + P : kcat SESI ---> ESI + P : kcat ISES ---> ISE + P : kcat [constants] Ks = 200, Ki = 0.004, kcat = 760? [responses] P = 1

9 [concentrations] E = 0.5 S = 400 [progress] rapid equilibrium [velocity] directory./anti/data extension txt mesh from 0 to 1.25 step variable I file anti2 conc. E = 0.5, S = 400 [output] directory./anti/output [settings] <Marquardt> interrupt = 100 [end]

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11 ; Dynafit script used for fitting of inhibition of steady-state activity by antimycin assuming activation of one monomer upon binding of one antimycin, without crossover [task] task = fit data = velocities model = activation of one monomer without crossover [mechanism] ; substrate and inhibitor equilibration in the dimer are the same as in the previous script ; catalytic steps: ES ---> E + P : kcat ; only one monomer is active SES ---> SE + P : kcat SEI ---> EI + P : kcat ; both monomers become active, but no crossover exists IES ---> IE + P : kcat SESI ---> ESI + P : kcat ISES ---> ISE + P : kcat [constants] Ks = 200, Ki = 0.004, kcat = 900?

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13 ; Dynafit script used for fitting of inhibition of steady-state activity by antimycin assuming crossover without activation of silent monomer [task] task = fit data = velocities model = crossover only [mechanism] ; substrate and inhibitor equilibration in the dimer are the same as in the previous script ; catalytic steps: ES ---> E + P : kcat ; both monomers are active without I SE ---> E + P : kcat SES ---> SE + P : kcat SES ---> ES + P : kcat SEI ---> EI + P : kcat IES ---> IE + P : kcat ESI ---> EI + P : kcat ; crossover allows activity of antimycin-bound monomer ISE ---> IE + P : kcat SESI ---> ESI + P : kcat ISES ---> ISE + P : kcat [constants] Ks = 200, Ki = 0.004, kcat = 450?

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15 ; Dynafit script used for fitting of inhibition of steady-state activity by antimycin assuming rapid intradimeric movement of inhibitor [task] task = fit data = velocities model = rapid intradimeric movement of inhibitor [mechanism] ; substrate and inhibitor equilibration in the dimer are the same as in the previous script ; catalytic steps: ES ---> E + P : kcat ; both monomers are assumed to be active SE ---> E + P : kcat SES ---> ES + P : kcat SES ---> SE + P : kcat SEI ---> EI + P : kcat IES ---> IE + P : kcat SESI ---> ESI + P : kcat ISES ---> ISE + P : kcat ; intradimeric movement: EI ---> IE : kintra ESI ---> IES : kintra SEI ---> ISE : kintra SESI ---> ISES : kintra IE ---> EI : kintra IES ---> ESI : kintra ISE ---> SEI : kintra ISES ---> SESI : kintra [constants] Ks = 200, Ki = 0.004, kcat = 490?, kintra = 5000

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17 ; Dynafit script used for determination of relative concentration of enzyme-inhibitor complexes [task] task = simulate data = equilibrate model = monomer ; E = enzyme ; I = inhibitor [mechanism] E + I <===> EI [constants] Ki = [concentrations] E = 1 [task] task = simulate data = equilibrate model = dimer [mechanism] E + I <===> EI EI + I <===> EI2 [constants] Ki =

18 [concentrations] E = 1 [equilibria] directory./ilicicolin/data extension txt mesh from 0 to 1.25 step 0.04 monitor E, EI, EI2 variable I file anteq conc. E = 1 [output] directory./anti/output [end]