Heme-peptide/protein ions and phosphorous ligands: search for site-specific addition reactions.
High-resolution Fourier transform ion cyclotron resonance mass spectrometry is employed to gain thorough kinetics and thermodynamics information on the reaction of free and ligated heme-type ions with selected ligands, with the aim of obtaining an insight into the coordination environment of the prosthetic group in a variety of biomolecular ions. Adopting a stepwise approach towards systems of increasing complexity, we examined the reactivity of free gaseous iron(III) protoporphyrin IX ions, Fe(III)-heme(+), of the charged species from microperoxidase-11 (MP11) (covalently peptide bound heme), and of the multiply charged ions from heme proteins, namely, cytochrome c (cyt c) and myoglobin (examples of noncovalently protein bound hemes). Among an array of test compounds allowed to react with Fe(III)-heme(+), OP(OMe)(3) and P(OMe)(3) proved to be similarly efficient ligands in the first addition step, yet displayed markedly distinct reactivity towards heme iron already engaged in axial coordination. The ease with which P(OMe)(3) acts as a second axial ligand is exploited to probe structural and conformational features of biomolecular ions. In this way, circumstantial evidence is gained of a folded conformation of +2 charge state ions from MP11 and an elongated one for the +3 charge state ions. Similarly, both the general reaction pattern and detailed kinetics and thermodynamics data point to a regiospecific addition reaction of P(OMe)(3) directed at the heme iron within multiply charged ions from cyt c. This unprecedented example of ion-molecule reaction which specifically involves a prosthetic group belonging to protein ions stands in contrast to the multiple, nonspecific interactions established by OP(OMe)(3) molecules with the protonated sites of multiply charged cyt c and apomyoglobin ions. This finding may develop and provide sensitive probes of the structure and bonding features of protein ions in the gas phase.
Crestoni
Maria Elisa
ME
Dipartimento Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università di Roma La Sapienza, P.le A. Moro 5, 00185, Rome, Italy. mariaelisa.crestoni@uniroma1.it
Fornarini
Simonetta
S
D000818:Animals; D005740:Gases / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006736:Horses; D008024:Ligands; D010455:Peptides / Q000737:chemistry*; D010758:Phosphorus / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
D005740:Gases; D008024:Ligands; D010455:Peptides; D011506:Proteins; D010758:Phosphorus; D006418:Heme