Unravelling the Intrinsic Features of NO Binding to Iron(II)- and Iron(III)-Hemes

Electrospray ionization of appropriate precursors is used to deliver [FeIII-heme]+ and [FeII-hemeH]+ ions as naked species in the gas phase where their ion chemistry has been examined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. In the naked, four-coordinate [FeII-hemeH]+ and [FeIII-heme]+ ions, the intrinsic reactivity of iron(II)- and iron(III)-hemes is revealed free from any influence due to axial ligand, counterion, or solvent effects. Ligand (L) addition and ligand transfer equilibria with a series of selected neutrals are attained when [FeII-hemeH]+, corresponding to protonated FeII-heme, is allowed to react in the FT-ICR cell. A Heme Cation Basicity (HCB) ladder for the various ligands toward [FeII-hemeH]+, corresponding to -ΔG° for the process [FeII-hemeH]+ + L f [FeII-hemeH(L)]+ and named HCBII, can thus be established. The so-obtained HCBII values are compared with the corresponding HCBIII values for [FeIII-heme]+. In spite of pronounced differences displayed by various ligands, NO shows a quite similar HCB of about 67 kJ mol-1 at 300 K toward both ions, estimated to correspond to a binding energy of 124 kJ mol-1. Density Functional Theory (DFT) computations confirm the experimental results, yielding very similar values of NO binding energies to [FeII-hemeH]+ and [FeIII-heme]+, equal to 140 and 144 kJ mol-1, respectively. The kinetic study of the NO association reaction supports the equilibrium HCB data and reveals that the two species share very close rate constant values both for the forward and for the reverse reaction. These gas phase results diverge markedly from the kinetics and thermodynamic behavior of NO binding to iron(II)- and iron(III)-heme proteins and model complexes in solution. The requisite of either a very labile or a vacant coordination site on iron for a facile addition of NO to occur, suggested to explain the bias for typically five-coordinate iron(II) species in solution, is fully supported by the present work.