Supplementary Information, Scheme and Figure Captions

SUPPLEMENTARY MATERIAL

Katherine A. de Villiers - Catherine H. Kaschula - Timothy J. Egan - Helder M. Marques

Speciation and structure of ferriprotoporphyrin IX in aqueous solution: spectroscopic and diffusion measurements demonstrate dimerization, but not m-oxo dimer formation

Scheme S1 The proposed relationship between the structure of the Fe(III)PPIX monomer and dimer and the pH-dependent behavior of aqueous Fe(III)PPIX.

Scheme S2 The standard porphyrin numbering system used for the Fe(III)porphyrin [53]

Fig. S1 A pair of H2O-Fe(III)PPIX molecules connected by two dummy atoms (Du) used to explore the structure of the hematin dimer.

Fig. S2 Spectra of Fe(III)PPIX solutions prepared as in Figure 1 (except concentration 1.9 ´ 10-5 M). The solid line shows the spectrum obtained when the stock Fe(III)PPIX solution was delivered using a Hamilton syringe, while the dotted line is that obtained when the same volume of the same solution was delivered with a calibrated plastic-tipped Gilson pipette.

Fig. S3 Effects of concentration on uv-vis spectra of solutions of aqueous Fe(III)PPIX. (A) Effects of concentration on the extinction coefficients of Fe(III)PPIX in the Soret band region, showing the presence of an isosbestic point at about 347 nm (indicated by a dotted vertical line). Arrows indicate the direction of change with increasing concentration. Conditions as in Fig. 1, except pH = 8.06 and Fe(III)PPIX concentration in the range 4 ´ 10-7 – 2 ´ 10-6 M. (B) A Beer’s law plot for A393. Here only the first four data points were fitted to a straight line, which was forced though the origin. Deviations below the line owing to Fe(III)PPIX self-association are already evident from 1 ´ 10-7 M and are very marked at higher concentrations (inset) where the data fall on a straight line of lower slope. Conditions in (B) as in (A), except that the pH was 7.69.

Fig. S4 Dependence of the Soret band maximum extinction coefficient (e393) of aqueous Fe(III)PPIX on concentration at (A) pH 7.43 and (B) pH 8.98. Solid lines are best fits to a dimerization equation (equation 2). Other conditions as in Fig. 1.

Fig. S5 The dependence of A610 on pH. Concentration of Fe(III)PPIX was 1 ´ 10-4 M, remaining conditions as in Fig. 1. The solid line is a best fit to the equation describing a single deprotonation. Residuals shown in the inset demonstrate a systematic deviation.

Fig. S6 (A) The 1H NMR spectrum of Fe(III)PPIX in d6-DMSO. The spectrum is similar to that reported by Budd et al. [45] for the same species and reported to be characteristic of a high-spin six-coordinate Fe(III)porphyrin. Tentative assignments based on Budd et al. are (i) methyl groups, (ii) vinyl a-CH and propionyl a-CH2, (iii) porphyrin meso H, (iv) propionic acid COOH group, (v) propionyl b-CH2 and (vi) cis and trans vinyl b-CH2. (B) The 1H NMR spectrum of the m-oxo dimer of Fe(III)PPIX induced in 10% v/v aqueous d6-pyridine in 0.1 M NaOD/D2O. The spectrum is similar to that reported for other m-oxo dimers of Fe(III)porphyrins reported by O’Keeffe et al. [27] with peaks assigned as (i) propionyl a-CH2, (ii) methyl groups and (iii) propionyl b-CH2. The broadened meso-H signals are expected to underlie the more prominent propionyl a-CH2 and methyl signals. The signal at about 9.5 ppm may arise from the vinyl a-CH groups which are not present in the derivatives described by O’Keeffe et al. In all NMR spectra sharp peaks indicated (*) are solvent signals.

Fig. S7 Effects of paramagnetic Fe(III)PPIX on the 1H NMR peak of H2O. (A) Shows the paramagnetic shift in the NMR signal of H2O owing to 5.48 ´ 10 -4 g×ml-1 aqueous Fe(III)PPIX at pH 8, while (B) shows that caused by 5.00 ´ 10-4 g×ml-1 of the m-oxo dimer of Fe(III)PPIX induced by 10% v/v aqueous pyridine in 0.1 M NaOH. The shifts are shown relative to water (pH 8) and 10% v/v aqueous pyridine in 0.1 M NaOH respectively (larger peak) and were determined at 303 K using a co-axial tube.

Fig. S8 Starting geometry used in the investigation of the structures of Fe(III)PPIX dimers. This structure represents the lowest energy structure obtained by MD/SA using Langevin dynamics to simulate the presence of solvent water molecules. It is 1.2 kcal mol-1 lower in energy than that shown in Fig. 8C.

Fig. S9 Structures of the Fe(III)PPIX dimer based on MD/SA simulations. The 25 lowest energy structures found for the H2O-Fe(III)PPIX dimer by MM calculations in which the structures were allowed to energy minimize freely with no physical connection between the pair of Fe(III)PPIX molecules. The number below each structure is its energy in kcal mol-1 relative to the lowest energy structure (marked as 0).