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The ligand substitutions that occur during the folding of ferrocytochrome c [Fe(II)cyt c] have been monitored by transient absorption spectroscopy. The folding reaction was triggered by photoinduced electron transfer to unfolded Fe(III)cyt c in guanidine hydrochloride (GuHCl) solutions. Assignments of ligation states were made by reference to the spectra of the imidazole and methionine adducts of N-acetylated microperoxidase 8. At pH 7, the heme in unfolded Fe(II)cyt c is ligated by native His18 and HisX (X = 26, 33) residues. The native Met80 ligand displaces HisX only in the last stages of folding. The ferroheme is predominantly five-coordinate in acidic solution; it remains five-coordinate until the native methionine binds the heme to give the folded protein (the rate of the methionine binding step is 16 +/- 5 s-1 at pH 5, 3.2 M GuHCl). The evidence suggests that the substitution of histidine by methionine is strongly coupled to backbone folding.

A novel one-step synthesis of the macrocyclic triserine trilactone scaffold of the siderophore enterobactin, which eliminates the β-lactonization step of N-tritylserine, is presented. The cyclization reaction is based on a stannoxane template and leads to an overall yield of ∼50%. This enables the practical functionalization of the trilactone by attaching chelating groups other than catecholamides. The conformational stability of the trilactone ring has been examined by high-resolution X-ray diffraction studies of the N-trityl intermediate: crystals grown from methylene chloride:methanol are orthorhombic, space group P2 1 2 1 2 1 with unit cell dimensions a ) 9.2495(5) Å, b ) 11.3584(1) Å, c ) 48.945(1) Å, V ) 5142.1(2) Å 3 , and Z ) 4. A hydroxypyridinonate analog of enterobactin, N,N′,N′′-tris [(3-hydroxy-1-methyl-2-oxo-(1H-pyridinyl)carbonyl]-4-cyclotriseryl trilactone (hopobactin), has been prepared by attachment of three 3-hydroxy-1-methyl-2(1H)-pyridinonate (3,2-HOPO) moieties to the triserine trilactone. This ligand represents the first enterobactin analog that retains the trilactone scaffold, but employs chelates other than catecholamides. Crystals of the chiral ferric complex grown from DMF:diethyl ether are monoclinic, space group P2 1 , with unit cell dimensions a ) 13.0366(9) Å, b ) 22.632(2) Å, c ) 27.130(2) Å, b ) 100.926(1)°, V ) 7860(1) Å 3 , and Z ) 8. The ∆ configuration of enterobactin metal complexes is also enforced in those of hopobactin and persists in aqueous or methanolic solution, as demonstrated by circular dichroism. The ferric hopobactin complex is the first reported chiral complex of hydroxypyridinonate ligands. The solution coordination chemistry of this new ligand and its iron(III) and iron(II) complexes have been studied by means of 1 H NMR, potentiometric, spectrophotometric, and voltammetric methods. The average protonation constant of the hopobactin free ligand (log K av ) 6.1) is typical of other 3-hydroxy-1-methyl-2-oxo-1H-pyridin-4-carboxamide ligands. The stability constants of the iron(III) complex formed with hopobactin (log β 110 ) 26.4) and with the tris(2-aminoethyl)amine-based analog, TRENHOPO, (log β 110 ) 26.7) are of the same order of magnitude, unlike the catecholamide-based species, where enterobactin (log β 110 ) 49) is 6 orders of magnitude more stable than TRENCAM (log β 110 ) 43.6). The stability enhancement reflects the specific predisposition by the triserine scaffold of the catecholamide binding units. In spite of a significantly lower affinity of 3,2-hydroxypyridinonates for iron(III) compared with the more basic catecholates, hopobactin is an extraordinarily powerful chelating agent under acidic conditions: No measurable dissociation is observed even in 1.0 M HCl. In contrast to enterobactin and its synthetic derivatives, the hopobactin ferric complex undergoes no sequential protonation above pH 1. The affinity of hopobactin and TRENHOPO for iron(III) relative to iron(II) results in strongly negative reduction potentials, -782 mV vs 0.01 M Ag + /Ag in CH...

The catechol siderophore analog K3[Ga(3)3], 4 (H2 3 = 2,3-dihydroxy-N,N‘-diisopropylterephthalamide), is D 3-symmetric in aqueous solution, and exists in two enantiomeric forms, Δ-4 and Λ-4. Variable temperature NMR experiments demonstrate that the inversion of the enantiomers of 4 in D2O is facile. The rate of inversion is independent of pH above pH 8. The mechanism is intramolecular. From line-shape analysis the free energy of activation ΔG ⧧ 298 = 67.4(9) kJ mol-1 in D2O at pD 12.1, with ΔH ⧧ = 58.5(6) kJ mol-1 and ΔS ⧧ = −0.030(9) kJ mol-1 K-1. Below pD 8 the rate of inversion for 4 is pD dependent and initially first order in [D+]. Potentiometric titrations reveal that 4 protonates in two one-proton steps with log K HML 3 = 4.66(4) and log K H 2 ML 3 = 3.99(7). In DMSO-d 6, formation of a tight contact ion pair between K+ and [Ga(3)3]3- ions increases the free energy barrier to inversion by ∼7 kJ mol-1. The complex K3[Ga(9)3], 10 (H2 9 = 2,3-dihydroxy-N-tert-butyl-N‘-benzylterephthalamide), was prepared to elucidate the mechanism of inversion by dynamic NMR spectroscopy, using the fact that 10 exists in two isomeric forms, cis-10 and trans-10, which are of C 3 and C 1 symmetry in solution. The ratio cis-10:trans-10 is 0.78(3) at ambient temperature in D2O or DMSO-d 6. Two processes are distinguishable on the NMR time scale in D2O or DMSO-d 6, cis- 10−trans- 10 isomerization and the inversion of the enantiomers of trans-10. Both processes proceed intramolecularly with T c = 295(1) K for Λ-trans-10 to Δ-trans-10 inversion and T c = 335(1) K for cis-10 to trans-10 isomerization in D2O at pD 9.5. The discrete exchange pattern of the tert-butyl resonances during inversion of trans-10 confirms that the reaction proceeds by a trigonal twist mechanism via a trigonal prismatic transition state. The free energy barriers to inversion are ΔG ⧧ 295 = 60 kJ mol-1 in D2O (pD 9.8) and ΔG ⧧ 327 = 67 kJ mol-1 in DMSO-d 6.

The amonabactins are a series of four bis(catecholate) siderophores isolated from the pathogenic organism, Aeromonas hydrophila. As tetradentate ligands, they cannot singly satisfy the octahedral coordination sphere of iron. The solution coordination chemistry of the amonabactins has been elucidated using potentiometric and spectrophotometric titrations, circular dichroism, and mass spectroscopy. They form 2:3 metal:ligand complexes at high pH and excess ligand. Their complexation behavior is essentially identical to one another, with log beta(230) = 86.3. At lower pH, they preferentially form a 1:1 bis(catecholato)bis(aqua) iron(III) species, with log beta(110) = 34.3. The 2:3 complexes show a very slight Delta preference in chirality at the metal center, while the 1:1 complexes are achiral. The biological implications of these properties are discussed.