Local Fe site structure in the tense-to-relaxed transition in carp deoxyhemoglobin: a XANES (x-ray absorption near edge structure) study.

Abstract

The Fe-site structure variation in the transition from the low-affinity tense (T) quaternary structure to the high-affinity relaxed (R) structure in carp deoxyhemoglobin was studied by analysis of multiple scattering resonances in the XANES (x-ray absorption near edge structure) spectra. High signal-to-noise XANES spectra were measured at the Frascati "wiggler" synchrotron radiation facility. We find that the forces on the Fe active site due to the change of quaternary protein conformation do not induce (i) variations >0.01 A in interatomic Fe-N distances, (ii) variations >0.1 A in the Fe displacement toward the heme plane, or (iii) the "doming" of the heme. The relevance of these results to the mechanism of protein control of ligand binding is discussed.Below pH 5.6 in the presence of allosteric effectors like inositol hexakisphosphate (Ins-P6), carp hemoglobin has a very low oxygen affinity and ligand binding is not cooperative (1)(2)(3)(4). This lack of cooperativity has been attributed (5) to the inhibition at acid pH of the allosteric transition from the tense (T) conformation of carp deoxyhemoglobin to the relaxed (R) conformation with ligand binding. On the other hand, the transition, with release of the ligand, from the R conformation of the ligated carp hemoglobin (HbCO and HbN3) to the T conformation of deoxyhemoglobin is precluded at alkaline pH. Therefore, carp deoxyhemoglobin presents the anomalous R conformation with high ligand affinity at alkaline pH that we will call here Rd. (Similarly, Td is the T quarternary state of carp deoxyhemoglobin at low pH.)Because allosteric effectors can induce the Td (low affinity) to Rd (high affinity) transition in carp deoxyhemoglobin, this protein has attracted much interest with the aim of investigating the correlation between the protein quaternary conformation and the local structure in the active site-i.e., the Fe position in the heme, which is a key point in the explanation of oxygen-binding cooperativity in hemoproteins (6,7). The interest is now addressed to the correlation between the variation of the quaternary structure (defined as the structure of the al/P82 interface) and the conformation of the immediate environment about the Fe site (which defines the protein tertiary structure) and the Fe coordination site structure.Diffraction studies to establish at the same time the protein conformation and local structure of the active site can be performed only on protein crystals. Spectroscopic methods that can be used to study the protein in solution, such as optical spectroscopy, resonance Raman spectroscopy, and magnetic susceptibility, are not direct probes of atomic positions.The structure of carp deoxyhemoglobin, as well as its variation from the low-to high-affinity form, has not been determined by crystallography. Therefore, there is a lack of direct experimental information on the variation of structure of this protein. Small changes have been observed in resonance Raman spectra between low-affinity deoxyhemoglobin and anomalous or patholog...

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