This journal is © The Royal Society of Chemistry 2000 S1
Supplementary material
“Dinuclear iron(III) complex with bridging urea anion: implications to urease mechanism”
Sergey V. Kryatov, Alexander Y. Nazarenko, Paul D. Robinson and Elena V. Rybak-Akimova*
Selected data for 2(ClO4)3: mass spectra: (ESMS+): m/z 2033 ([(2)2(ClO4)5]+, 15%), 965 ([2(ClO4)2]+, 100%); (FAB+): m/z 965 ([2(ClO4)2]+, 100%), 866 ([1(ClO4)]+, 40%); exact mass FAB+ determination: calc. for [2(ClO4)2]+m/z 965.0926, found m/z 965.0959; calc. for [(15N2-2)(ClO4)2]+m/z 967.0866, found m/z 967.0878; UV-vis: max (MeCN)/nm (): 318 (13800), 501 (1050), 690 (135); 1H NMR (300 MHz, 25oC, CD3CN): 30, 28, 26, 22, 19 ( pyr or CH2), 17.3, 16.2, 12.2, 11.9 ( pyr), 10.8 ( pyr or CH2), 7.82, 7.22, 6.86, 6.57 ( pyr). The 1H NMR assignment to different groups of protons in the tpa ligands was made in analogy to other [Fe2(-O)(-L)(tpa)2]n+ species.7 The protons of the -OC(NH2)NH ligand are probably exchanging with the trace water in the CD3CN solution ( 2.2 under our conditions), and so do not appear in the spectrum separately.
Selected experimental conditions: Concentrations used for kinetic study: [1] = 0.5-1 mM, [H2O] = 0.05 - 0.6 M, [urea] = 4 - 60 mM. Rate constants kobs are wavelength independent in the region 400-800 nm.
Fig. S1 Kinetic data in double reciprocal (Lineweaver-Burk) coordinates. Linear fits at different [H2O], M: 0.05 (1), 0.1 (2), 0.2 (3), 0.3 (4), 0.4 (5), 0.6 (6). (See Table S1 for the values of [urea], [H2O], and kobs).
Treatment of the kinetic data. The Lineweaver-Burk plot (Fig. S1) is reminiscent of mixed (noncompetetive) inhibition in classic enzymatic kinetics, suggesting that there are at least two preeqilibria involving urea and water before the rate limiting step. Kinetic models similar to mixed inhibition were considered. (It should be kept in mind that the enzymatic kinetic treatment is applicable directly only to catalytic reactions, which is not our case).
Model A:
Starting from the definitions and assumptions:
[1]T = [1] + [1a] + [1b] + [1c] (S.A1)
Kb = (S.A2)
Kc = (S.A3)
d[2]/dt = -d[1]T/dt = kobs[1]T = k2[1a] (S.A4)
d[1a]/dt = k1[urea][1] – [1a] (k-1 + k2) (S.A5)
and using a derivation similar to that for mixed inhibition in classic enzymatic kinetics [D.Voet, J.G.Voet, Biochemistry, 2nd Edition, John Wiley, 1995, p.359] we obtain:
kobs = (S.A6)
= + (S.A7)
Model B:
1 + CO(NH2)2⇄ 1a + H2O, fast; K1 = (S.B1)
1a + H2O ⇄ 1b, fast; K2 = (S.B2)
1a 2, rate limiting step;(S.B3)
[1] + [1a] + [1b] = [1]T(S.B4)
Combination of (S.B4) with (S.B1) and (S.B2) yields:
[1a] = [1]T(S.B5)
Rate of the overall reaction is:
d[2]/dt = - d[1]T/dt(S.B6)
d[1]T/dt = - k3[1a] = -[1]T(S.B7)
that would correspond to a single exponential decay of [1]T with:
kobs = (S.B8)
= + (S.B9)
Problems of applying Models A and B. Attempts to fit the experimental data with either Model A or Model B were rather unsuccessful, because they do not fit the dependence of kobs on [H2O]. According to Models A and B, the slope of kinetic plots in double reciprocal (Lineweaver-Burk) coordinates should be a linear function of [H2O], as follows from equations S.A7 and S.B9. (See also C.H.Suelter, A Practical Guide to Enzymology, John Wiley, 1985, p.248). The experimental data presented in Fig. S2 show that this is not the case.
Fig. S2 Slopes of Lineweaver-Burk kinetic plots versus [H2O] (see Fig. S1) and their successful fit with a second order polynomial function, LB-slope = 0.204[H2O]2 + 0.053[H2O].
Also, Models A and B are in contradiction with the chemistry of the system. Complex 1 is hydrolytically stable under the concentration conditions used (S.V.Kryatov, E.V.Rybak-Akimova, unpublished results). Thus, equilibrium S.A2 in Model 2 can not influence the overall kinetics of the 12 transformation. The same reasoning is applicable to equilibrium S.B2 in Model B.
Model C. (Shown in Scheme 1, main text).
Additional assumptions of this model are:
[1], [1a] > [1b] (S.C1)
[1]T = [1] + [1a] (S.C2)
[H2O], [urea] > [1]T(S.C3)
K1 = (S.C4)
Also, it is assumed that step 3 is irreversible under the concentration conditions used:
d[2]/dt = -d[1]T/dt = kobs[1]T = k3[1b] (S.C5)
Speciation in the preequilibrium mixture of 1, 1a, urea and water (step 1) yields:
[1a] = [1]T(S.C6)
Assuming steady state condition for 1b:
d[1b]/dt = k2[1a] - k3[1b] – k-2[1b][H2O] = 0(S.C7)
[1b] = [1a] = [1]T(S.C8)
Rate of the overall reaction is:
d[1]T/dt = - k3[1b] = -[1]T(S.C9)
corresponding to a single exponential decay of [1]T with:
kobs = (S.C10)
= + (S.C11)
Table S1. Raw kinetic data, their fit by the kinetic equation C.S10 (equation 3, main text) and the final equilibrium yield of complex 2 in preequilibrium 1 (main text).
[urea], M / [H2O], M / kobs, s-1 / kcalc, s-1 / Relative deviation D,|kobs - kcalc|/kobs / Yield of
Complex 2
0.02975 / 0.05 / 1.8 / 1.721 / 0.044 / 0.9998
0.01775 / 0.05 / 1.62 / 1.554 / 0.041 / 0.9997
0.01055 / 0.05 / 1.37 / 1.335 / 0.025 / 0.9996
0.00623 / 0.05 / 1.12 / 1.076 / 0.039 / 0.9993
0.003638 / 0.05 / 0.822 / 0.804 / 0.021 / 0.9989
0.02975 / 0.1 / 1.14 / 1.225 / 0.074 / 0.9994
0.01775 / 0.1 / 0.96 / 1.033 / 0.076 / 0.9991
0.01055 / 0.1 / 0.77 / 0.817 / 0.061 / 0.9985
0.00623 / 0.1 / 0.67 / 0.602 / 0.102 / 0.9975
0.003638 / 0.1 / 0.46 / 0.413 / 0.103 / 0.9957
0.02975 / 0.2 / 0.7 / 0.712 / 0.017 / 0.9979
0.01775 / 0.2 / 0.526 / 0.551 / 0.048 / 0.9965
0.01055 / 0.2 / 0.376 / 0.399 / 0.061 / 0.9942
0.00623 / 0.2 / 0.25 / 0.271 / 0.085 / 0.9902
0.003638 / 0.2 / 0.163 / 0.174 / 0.068 / 0.9833
0.02975 / 0.3 / 0.478 / 0.465 / 0.027 / 0.9953
0.01775 / 0.3 / 0.338 / 0.342 / 0.012 / 0.9922
0.01055 / 0.3 / 0.231 / 0.236 / 0.023 / 0.9870
0.00623 / 0.3 / 0.152 / 0.155 / 0.018 / 0.9782
0.003638 / 0.3 / 0.096 / 0.097 / 0.006 / 0.9633
0.02975 / 0.4 / 0.338 / 0.327 / 0.031 / 0.9917
0.01775 / 0.4 / 0.237 / 0.233 / 0.018 / 0.9863
0.01055 / 0.4 / 0.157 / 0.156 / 0.004 / 0.9772
0.00623 / 0.4 / 0.101 / 0.1 / 0.009 / 0.9619
0.02975 / 0.6 / 0.209 / 0.188 / 0.102 / 0.9817
0.01775 / 0.6 / 0.136 / 0.128 / 0.061 / 0.9697
Davr = 0.045