Protonated Heme

Abstract

The ions formally corresponding to protonated heme [Fe(II)-hemeH](+) have been obtained by collision-induced dissociation from the electrospray ionization of microperoxidase (MP11) and their gas-phase chemistry has been studied by FTICR mass spectrometry. H/D-exchange reactions, used as a tool to gain information on the protonation sites in polyfunctional molecules, show that labile hydrogens pertain to the propionyl substituents at the periphery of the protoporphyrin IX. Several conceivable isomers for protonated heme have been evaluated by density functional theory. The most stable among the species investigated is the one corresponding to protonation at the beta carbon atom of a vinyl group, yielding a proton affinity (PA) value for [Fe(II)-heme] of 1220 kJ mol(-1). This high PA is consistent with the inertness of the hydrogen atoms at the protonation site towards H/D exchange with ND(3) and CD(3)CO(2)D. Peculiar features of this [Fe(II)-hemeH](+) isomer emerge by analysis of its electronic structure, showing that the vinyl group undergoing formal protonation has gained significant radical character due to electron transfer from the metal center. As a consequence, the iron atom acquires partial iron(III) character and none of the two formal descriptions [Fe(II)-hemeH(+)] and [Fe(III)-hemeH(.)](+) alone may adequately illustrate the protonated heme ion. In agreement with this description, the reactivity of protonated heme presents dual facets, resembling iron(III) in some aspects and iron(II) in others. On the one hand, protonated heme behaves like [Fe(III)-heme](+) ions in H/D-exchange reactions. On the other, it shows markedly decreased reactivity towards the addition of ligands with the notable exception of NO, in line with the high affinity shown by iron(II) complexes towards this molecule, NO, of key biological role.

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

“…10 The so-obtained description of [Fe II -hemeH(NO)] + is consistent with that of an iron(II)-heme bound to NO. A binding energy of 140 kJ mol -1 is calculated with respect to dissociation into triplet [Fe II -hemeH] + and 2 NO.…”

“…When [Fe II -hemeH] + ions extracted from the ESI source and isolated in the FT-ICR cell are exposed to a stationary concentration of selected neutral ligands (L), an addition reaction may be observed, leading to [Fe II -hemeH(L)] + . For the addition reaction between [Fe II -hemeH] + and ligands such as NO and (C 2 H 5 O) 3 P the second order rate constants of 0.33 × 10 -10 cm 3 molecule -1 s -1 and 4.0 × 10 -10 cm 3 molecule -1 s -1 , respectively, have been reported, as determined at 300 K. 10 In the present, thorough study, a vast array of compounds were tested and many were found unreactive, including biologically active diatomics (O 2 and CO), nitrogen bases (NH 3 , CH 3 NH 2 , CH 3 CN), carboxylic acids and derivatives (acetic acid, CH 3 C(O)N(CH 3 ) 2 ), alcohols and ethers (CH 3 OH, tetrahydrofuran, 2-methoxypropene), ketones ((CH 3 ) 2 CO), and sulfides ((CH 3 ) 2 S, (CH 3 ) 2 S 2 )). In relatively few cases, adduct ions are formed and the relevant thermodynamic features of the process are now reported.…”

“…21 This choice was justified by noting that the electronic properties of the complex are expected to be insensitive to the presence and nature of peripheral substituents. 21c,22 A similar simplified model was adopted in characterizing the structural, electronic, and spectroscopic features of iron porphyrin-NO 10 In the present context, the binding energy of NO to both [Fe II -hemeH] + and [Fe III -heme] + , the species studied for NO addition in the gas phase experiments, has been evaluated using DFT. To this end the electronic energies for all species partaking in eqs 5 and 6 have been calculated for the optimized structures in the electronic ground state.…”

Intrinsic Properties of Nitric Oxide Binding to Ferrous and Ferric Hemes

“…This novel complex has been thoroughly characterized both by its gas phase reactivity and by computations searching for plausible structures. 23 By DFT calculations, the relative stability of various isomers holding the additional proton on different sites of the porphyrin ligand, on its substituents and on the metal atom itself has been assessed. The most stable isomer is the one corresponding to protonation on one of the peripheral vinyl groups and the optimized structure is shown in Figure 1.…”

“…In the study of association reactions either the reactant ion or the formed ion-neutral complex may be isolated and their reaction kinetics evaluated at varying neutral pressure. In this way the rate constants for the forward and reverse reaction (k f and k r , respectively) may be evaluated ( 23,25 Their ratio, yielding the reaction equilibrium constant, has been compared with the equilibrium constant value derived from the steady ion abundance ratio observed at long reaction time, the two values nicely matching each other. Exemplary kinetic plots are shown in Figure 2 .…”