Comparison of the magnetic properties of deoxy- and photodissociated myoglobin.

Abstract

The magnetic susceptibility of photodissociated carbon monoxy myoglobin has been measured over the temperature range from 1.7 to 25 K at 10 and 50 kG with a superconducting susceptometer. The spin and the crystal field parameters of the iron ion were extracted by a spin Hamiltonian approach. Under equivalent conditions the magnetic susceptibility of deoxy myoglobin was measured. In both experiments the CO-bound protein was used as a diamagnetic reference. Above about 5 K the metastable photolysed state and the equilibrium deoxy form of myoglobin are magnetically indistinguishable and can be fitted with S = 2 and g = 2. The transition from spin 0 to spin 2 and the conformational changes known to accompany the electronic change thus also occur after photolysis at low temperature. At temperatures below 5 K, differences become apparent, indicating a somewhat smaller zero-field splitting in the photoproduct as compared to the ligand-free state at equilibrium. In qualitative agreement with observations made by other techniques, the data imply that even at 1.7 K substantial structural relaxation occurs in the heme region of myoglobin after photodissociation. The results are important for the interpretation of the ligand binding kinetics after flash photolysis at low temperature and contribute to the understanding of the relationship between electronic structure and function in heme proteins. Heme proteins perform a wide variety of functions, from oxygen storage to detoxification. Since they all share the same prosthetic group, a detailed understanding of the function of one heme protein can provide insight into a broader field. The binding of small ligands such as dioxygen and CO to ferrous Mb and Hb is probably the simplest such biological process and it has been studied with a variety of tools (1-4). Flash photolysis experiments over a wide range of time and temperature have established that the controlling reaction in ligand binding occurs at the heme group and involves the heme iron (5, 6). In the discussion of the crucial association and dissociation step it is generally assumed that the ligandfree (deoxy) form of the ferrous heme is in the high-spin state (S = 2) while the ligand-bound form is in the low-spin state (S = 0). Consequently, association and dissociation of 02 and CO are assumed to be accompanied by a spin change of 2.The assignment of the spin states is based on the pioneering magnetic susceptibility measurements of Pauling and Coryell (7) on Hb and related proteins. MbCO was shown to be diamagnetic by Theorell and Ehrenberg (8). The magnetic properties of deoxy Hb and Mb have been studied by Nakano et al. (9). In addition to verifying the spin state they characterized the electronic fine structure of the iron in these heme proteins.Ligand binding not only modifies the electronic structure of the heme iron but also induces distinct changes in the conformation of the heme and its environment (10). X-ray crystallography shows that in deoxy Mb (11) the iron ion lies out of the mean heme plane by...

Metastable photoproducts from carbon monoxide myoglobin.

“…The spectra ofthe photoproduct at low temperatures reported here are characteristic of high-spin five-coordinate hemes, consistent with other studies that have also shown that the iron atom in the photoproduct is high spin (S=2) (11). We conclude that the photoproduct generated at very low temperatures (1.6 and 4 K) has a high-spin heme that is slightly perturbed compared to the heme in the equilibrium deoxy preparation.…”

Integer-spin electron paramagnetic resonance of iron proteins

“…13. The parameter set, which now invokes a spread in parameters D and E, is compatible with magnetic susceptibility (40) and far-infrared (22) results. However, the Mb spectra are similar to those of the hexaquo Fe+2 and FeTPP with the trough in dx'"/dB for Mb occurring 5 mT higher and being approximately a factor of three weaker than in the FeSO, spectra of Fig.…”

Global Mapping of Structural Solutions Provided by the Extended X-ray Absorption Fine Structure ab Initio Code FEFF 6.01: Structure of the Cryogenic Photoproduct of the Myoglobin−Carbon Monoxide Complex

“…The exciting features of this structure were (1) the ligand was located in the heme pocket 3-4 Å from the iron atom essentially parallel to the heme and (2) the proximal histidine-iron coordinate had moved close the deoxy structure but was essentially relaxed only 75% toward the deoxy coordinates. This provided reasonable structural interpretations for both the infrared signature of the B states, which are close to the free gas value of the CO stretching frequency (Alben et al, 1982;Ansari et al, 1987), and the high-spin (S ) 2), outof-plane spectroscopic assignment for the iron atom based on optical, Mossbauer, magnetic susceptibility, and X-ray absorption spectroscopy (XAS) data (Chance, 1993;Iizuka et al, 1974;Marcolin et al, 1979;Miller & Chance, 1995;Roder et al, 1984;Spartalian et al, 1976). In addition, recent time-resolved photoselection spectroscopy experiments have provided additional details about the CO ligand geometry in the bound state and its trajectory upon photolysis (Lim et al, 1995).…”