Probing the Electronic Structure of the Hemoglobin Active Center in Physiological Solutions

Abstract

Soft-x-ray absorption spectroscopy at the L 2;3 edge of the iron center in bovine hemoglobin and hemin under physiological conditions is reported for the first time. Spectra of the same compounds in solid form are presented for comparison. Striking differences in the electronic structure of the metalloporphyrin are observed between the liquid and solid compounds. We unambiguously show that hemoglobin and hemin are in a high-spin ferric state in solution, and that the 2p spin-orbit coupling decreases for hemin compared to the hemoglobin, while this is not the case in solids. The spectra were simulated using ligand field multiplet theory, in good agreement with the experiment, allowing quantification of the amount of charge transfer between the porphyrin and Fe 3þ ion in hemoglobin and in hemin. DOI: 10.1103/PhysRevLett.102.068103 PACS numbers: 87.64.Àt, 87.85.jc, 87.15.Àv, 87.80.Dj Hemoglobin (Hb) is the respiratory protein of the red blood cells which carries oxygen from the lungs to the tissues in order to maintain the viability of cells. The active center of Hb is the heme group [ Fig. 1(a), inset], which is located at the center of the protein and consists of a flat porphyrin ring molecule with an Fe 2þ or Fe 3þ ion center. Hemin is the salt form of the heme group with a Cl coaxial ligand instead of the imidazole group of histidine in Hb [ Fig. 1(b), inset]. Describing and understanding the process by which heme proteins discriminate between ligands, in particular, diatomics, that bind to the iron atom has been the subject of intense studies for several decades [1][2][3][4][5][6].The study of the active metal center by optical spectroscopy is difficult because the intense ! Ã porphyrin transitions obscure the (forbidden) transitions to d orbitals of the metal, which are involved in the binding process. To circumvent this problem, techniques such as nuclear magnetic resonance (NMR) [7], electron paramagnetic resonance (EPR) [8], and Mössbauer spectroscopy [9] have been employed. Although NMR and EPR delivered detailed pictures about the splitting of the d orbitals, they do not provide direct information about the nature of the bonding between the Fe ion and the ligand. Moreover, neither EPR nor Mössbauer spectroscopy have to our knowledge been applied to heme proteins in physiological solutions.X-ray absorption spectroscopy (XAS) is ideal for probing the electronic and geometric structure of the active site. Hard-x-ray absorption spectroscopy studies have been carried out at the K edge of iron [10,11]. While much insight was gained about the local geometric structure around the iron atom, these do not deliver information about the electronic structure. For this purpose, soft-x-ray L 2;3 edge spectroscopy (due to 2p 1=2;3=2 ! 3d transitions, respectively) for first row (3d) transition metals is much more appropriate, since: (a) The smaller intrinsic core hole lifetime width (0.5 eV) of p orbitals results in sharper spectral features than K edge (1s ! 3d) ones; (b) 2p 1=2;3=2 ! 3d transitions are dipole-allo...

Retracted Article: On the enzymatic activity of catalase: an iron L-edge X-ray absorption study of the active centre

“…The L 3 -edge feature exhibits a doublet in both cases, but the low energy band is almost three times weaker relative to the main peak in catalase than in metHb, while the energy difference between the two peaks is identical. As already discussed, 2,19 the first component is due to transitions from the p-core orbital to the valence d xy , d yz and d xz orbitals, while the second is due to transitions to the d x 2 Ày 2 and d z 2 orbitals. The similar energy splitting in both systems shows that, just as for metHb, 2 catalase is in a high spin (HS) state.…”

“…As already discussed, 2,19 the first component is due to transitions from the p-core orbital to the valence d xy , d yz and d xz orbitals, while the second is due to transitions to the d x 2 Ày 2 and d z 2 orbitals. The similar energy splitting in both systems shows that, just as for metHb, 2 catalase is in a high spin (HS) state. The decreased intensity of the low energy feature may suggest a larger occupancy of the d xy , d yz and d xz orbitals in catalase, but as shown below, its origin is different.…”

“…Details about the set up have been discussed in detail before. 2,23 The sample temperature was varied from 5 up to 30 1C. Higher temperatures (up to 37 1C) showed no changes in the spectrum.…”

“…This activity has one of the highest turnover numbers of all enzymes: one molecule of catalase can convert up to 10 6 hydrogen peroxide molecules to water and oxygen per second. 1 Surprisingly, metHb, which is in a high spin (HS) state, 2 carries out this function with a much lower efficiency. This is also the case with myoglobin (Mb), which is a monomer that is structurally very close to Hb monomers.…”

“…22 We recently extended these studies to proteins in physiological solutions and recorded the Fe L-edge spectra of haemoglobin and hemin. 2 The issue of radiation damage was critical, and the solutions had to be renewed by continuous flowing (B1 l min À1 ). This first experiment opened the way to a systematic investigation of the electronic structure of the active centre of haem proteins in physiological solutions using Fe L-edge spectroscopy.…”