Metastable intermediates in myoglobin at low pH.

Abstract

Resonance Raman and optical absorption spectra of ligand-free (deoxy) myoglobin and CO-bound myoglobin (MbCO) at pH 2.6 have been measured by using continuous-flow/rapid-mixing techniques. The spectra of deoxy myoglobin at low pH within 6 ms of the pH drop demonstrate that the iron-histidine bond has been ruptured but that the heme is still five-coordinate. Comparison with data from model complexes indicates that a weak-field ligand, such as a water molecule, is coordinated at the fifth position. The Raman spectrum of MbCO at low pH has an Fe-CO stretching mode that is characteristic of a six-coordinate heme with an unhindered Fe-CO moiety. Immediately following the pH drop in this case, there is no indication that the iron-proximal histidine bond is broken. Three different structural changes are detected at low pH: (i) the iron-proximal histidine (F8) bond in ligandfree myoglobin is broken and replaced by a weak-field ligand, (ih) the distal pocket in MbCO is opened, and (iii) protein constraints on the heme group in MbCO are relaxed. Previous conclusions that the kinetics of CO-binding in hemoproteins at low pH is modified by rupturing the iron-proximal histidine bond are supported by these new results which, however, demand a more complete reevaluation of the phenomenon.Since heme proteins carry out diverse biological functions, a great deal of effort has gone into determining the molecular basis for their properties. Myoglobin (Mb) is one of the simplest heme proteins and has been extensively studied in order to understand the factors that control the binding and release of oxygen, its physiological ligand, as well as CO, which is a convenient oxygen substitute for laboratory studies (1, 2). In several reports, the binding of CO to Mb (3,4) and other heme proteins (5, 6) has been monitored after a rapid drop in pH. For most Mbs and monomeric hemoglobins (Hbs) (3, 4) and for human tetrameric Hb (5), it was found that at low pH (-2.5) a large increase in the CO-binding rate occurs. This was interpreted as a consequence of protonation of the proximal histidine and the associated rupture of the iron-histidine bond, thereby allowing the iron atom to adopt an in-plane position and thus lower the barrier for CO binding. The rupture of the iron-histidine bond was supported by the absorption spectra, which, in the case of the ferrous ligand-free protein at low pH, are similar to those of fourcoordinate model compounds (4).Resonance Raman scattering is a well-suited technique to determine ligand coordination and the structure of the active site in heme proteins. Modes in the high-frequency region are known to be sensitive to axial coordination, porphyrin macrocycle core size, and heme doming (7). Axial ligand modes are often present in the resonance Raman spectrum and may be used to determine the heme coordination and the structure of the ligands (8,9). In addition, the properties of the iron-histidine bond may be monitored, since the ironhistidine stretching mode is present in five-coordinate ferrous hemes (1...

Effects of urea and acetic acid on the heme axial ligation structure of ferric myoglobin at very acidic pH

“…These spectral variations are indicative of the conversion of the aqua six-coordinate high-spin heme (6cHS) native form of the protein [denoted as 6cHS(1)] to a five-coordinated high-spin (5cHS) species. In fact, at acid pH the length of the Fe-proximal histidine bond increases [65], and breaks in ferric Mb below pH 4, whereas a water molecule binds to the iron atom [64–66]. Accordingly, the corresponding RR spectrum in the high frequency region (Figure 1B) changes from the core size marker bands typical of a 6cHS heme (ν 3 at 1483 cm −1 and ν 2 at 1563 cm −1 ) [67] to that of a 5cHS heme with a water molecule coordinated to the heme iron atom [64].…”

Redox-controlled Ligand Exchange of the Heme in the CO-sensing Transcriptional Activator CooA

“…1 and 5). On the other hand, the electronic absorption spectrum of ferrous H77Y CooA showed the Soret band and a single peak in the Q-band region at 424.0 and 558.5 nm, respectively, which resembled the spectrum of deoxymyoglobin (41) and was typical of five-coordinate, high-spin ferrous hemeproteins. Shelver et al (16) have reported that H77Y CooA cannot be stably reduced by dithionite (although the data are not shown), which is inconsistent with our result.…”

Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

“…In particular, the conformation sensitive interaction of the imidazole side chain of the distal histidine greatly impacts the frequency [66]. In an aqueous solution of COHbA at pH 6.5 with glycerol added at a 12.5% (vol/vol) level, the frequency of the Fe-CO band is ~405 cm −1 which corresponds to a distal heme pocket conformation in which the imidazole side chain is in the pocket, as observed under most ambient conditions in the pH range from 6 to 8 [67]. When this same solution of COHbA is encapsulated in an RM at an added volume that matches that which is used to create a ω 0 20x sample for a glycerol free aqueous solvent, there appears a strong second peak at 492 cm −1 .…”