Supplementary material for Lombardi et al. (2000) Proc. Natl. Acad. Sci. USA 97 (22), 11922–11927.

Metal-Binding Experiments.

The binding affinity of Co(II) to miniaturized electron transfer protein (METP) peptide was measured by CoNO3·titration of the reduced peptide at 7.54 ´ 10–4 M concentration, in 10 mM phosphate buffer, pH 7.5, under anaerobic conditions. The absorption spectra in the 300- to 750-nm region were recorded. The change in the spectrum in this region on addition of CoNO3 is because of the metal complex formation CoPn, where Co, P, and n indicate the cobalt ion, the METP peptide, and the stoichiometry of the interaction, respectively. The absorbance A at a given wavelength is related to the metal complex concentration through the Lambert–Beer equation:

A = e[CoPn]l,                                                                                                     [1]

where e is the molar extinction coefficient (M–1·cm–1), and l is the path length (in centimeters).

The dissociation constant and the stoichiometry of the interaction were obtained by fitting the observed absorbances to a binding isotherm, for the equilibrium:

1/nCoPn « P + 1/nCo KdCo = [Co]f1/n[P]f/[CoPn]1/n,                                                             [2]

in which [Co]f, [P]f, [CoPn] are the concentrations of free cobalt, free peptide (in monomeric units), and complex, respectively.

The total peptide concentration [P]t is:

[P]t = [P]f + [P]b [P]f = [P]t – [P]b,                                                                                          [3]

where [P]b is the molar concentration of the bound peptide.

The total cobalt concentration [Co]t at each titration point is:

[Co]t = [Co]f + [Co]b [Co]f = [Co]t – [Co]b,                                                                           [4]

where [Co]b is the molar concentration of the bound cobalt ion.

Further, from the stoichiometry of the reaction:

[P]b = n [CoPn] [Co]b = [CoPn]                                                                                               [5]

Introducing Eq. 5 into Eqs. 3 and 4:

[P]f = [P]t – n [CoPn]                                                                                                               [6]

[Co]f = [Co]t – [CoPn]                                                                                                             [7]

Substitution of Eqs. 6 and 7 into Eq. 2 gives:

Kd = ([Co]t – [CoPn])1/n ([P]t – n [CoPn])/[CoPn]1/n                                                              [8]

Introducing into Eq. 8

R = [Co]t/[P]t and n [CoPn]/[P]t = (A – Ao)/(A¥ – Ao),                                                          [9]

where Ao, A, and A¥ are the absorbances at a given wavelength in the absence, in the presence, and at saturation of the cobalt ions. Solving R vs. (A – Ao)/(A¥ – Ao), it gives:

[R] = (1/n)Kdn(A – Ao)/(A¥ – Ao)/([P]t – [P]t(A – Ao)/(A¥ – Ao))n

           + (1/n) (A – Ao)/(A¥ – Ao).                                                                                        [10]

Best fit of the data [by using the software package KALEIDAGRAPH (Synergy Software, Reading, PA)], with [P]t = 7.5 ´ 10–4 M, gave the stoichiometry of the interaction n = 2, and KdCo = 53.5 (± 2.8) mM.

The affinity of Zn(II) was estimated by competition experiments on the Co(II) complex. Aliquots of ZnCl2·6H2O aqueous solution (1.9 ´ 10–2 M) were titrated into a solution containing the reduced peptide at 5.71 ´ 10–4 M concentration and 3-fold molar excess of Co(II), at pH 7.5. The bleaching of the absorption spectrum was monitored.

The relative affinity of the interaction was obtained by fitting the data to the equilibrium:

CoP2 + Zn2+·6H2O « ZnP2 + Co2+·6H2O Kex = [ZnP2][Co]f / [CoP2] [Zn]f,                       [11]

where [ZnP2], [CoP2], [Co]f, [Zn]f, are the concentrations of Zn(II) complex, Co(II) complex, free cobalt, and free zinc ions, respectively.

Introducing into Eq. 11

R = [Zn]t/[P]t and 2 [CoP2]/[P]t = (A – A¥ )/(Ao – A¥ ),                                                     [12]

where A, Ao, and A¥ are the absorbances at a given wavelength, in the absence, in the presence, and at saturation of the zinc ions. Solving R vs. (A – A¥ )/(Ao – A¥ ), it gives:

[R] = [1 – (A – A¥ ) / (Ao – A¥ )] / Kex (A – A¥ ) / (Ao – A¥ ) ([Co]t/[P]t – 0.5 (A – A¥ ) /

       (Ao – A¥ )) + (0.5 – 0.5(A – A¥ )/(Ao – A¥ ).                                                            [13]

The best fit of the data, with [P]t = 5.71 ´ 10–4 M and [Co]t = 1.65 ´ 10–3 M, gave a value for the exchange reaction constant Kex = 393 ± 1. It was possible to estimate the dissociation constant for the Zn(II) complex from the dissociation constant for the equilibrium [1] KdissCo = 53.5 (± 2.8) mM, and (Kex)1/2 = KdissCo/KdissZn ; KdissZn = 2.7 (± 0.1) mM.