Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures.

Abstract

We have studied the infrared spectra of the bound and photodissociated states ofMb-"2CO and Mb-'3CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-'2CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-'. The low-temperature photodissociated state (Mb*) shows three lines (BO, B1, B2) at 2144, 2131, and 2119 cm-' for "2CO. The absorbances of the three lines depend on temperature. Bo is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb* have near-infrared, EPR, and Mossbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb* and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.The reversible binding of CO to the storage protein Mb can be studied with flash photolysis (1). Experiments in which the Soret line was monitored demonstrate that the binding process involves a number ofsteps (2, 3). Here we show that monitoring the CO stretching vibration reveals additional features of the protein's interior.The active center of Mb, the heme group, is embedded in the protein (Fig. 1) (4) and the ligand binds at the central heme iron. In flash photolysis, the bound-state MbCO is photodissociated. Below 200 K the CO cannot leave the heme pocket and rebinds from there. Two states are involved in low-temperature recombination: state A, in which the CO is bound, the heme is nearly planar, and the iron atom has spin 0; and state B, in which the CO is photodissociated from the heme iron and remains in the protein pocket and the iron has spin 2. At low temperatures, the rebinding process B to A is not exponential in time. We have explained this observation by postulating the existence of conformational substates (2, 5). At low temperatures each Mb molecule is frozen into a particular substate with a specific barrier height for rebinding. From 180 to 80 K the transition occurs by an over-the-barrier Arrhenius process; below 60 K, quantum mechanical tunneling dominates (6, 7). State B (Mb*) has been studied in MbCO and CoMbCO by near-infrared (8, 9), EPR (10, 11), and Mbssbauer (12) Pentex (Kankakee, IL) was dissolved in 70% (vol/vol) glycerol in water buffered to pH 7 with 0.1 M phosphate. The sample was stirred under a CO atmosphere for several hours, reduced with sodium dithionite, and stirred for s...

Influence of Distal Residue B10 on CO Dynamics in Myoglobin and Neuroglobin

“…They are found trapped at the primary docking site B on top of pyrrole C [25,27,30]. Opposite orientations of the ligands in the B site of A 1 molecules are represented by the photoproduct bands at 2,119 and 2,131 cm −1 [21,46]. In the A 3 conformation, ligands at site B also give rise to a doublet of bands, but only the band at 2,149 cm −1 is spectrally separated (Fig.…”

“…The strong infrared absorption of the bound CO ligand is highly sensitive to the local electric field at the binding site and indicates structural heterogeneity. Multiple active-site conformations had been inferred from the CO stretching spectrum of wild-type MbCO [46] long before their individual structures were revealed by X-ray crystallography [47,48]. The CO ligand can be photodissociated from the iron at cryogenic temperatures and can migrate to trapping sites B, C, and D, where it gives rise to characteristic stretching bands [21,37].…”

A unified model of protein dynamics

“…Two of the taxonomic substates in Mb, A 0 and A 1 , have been studied in detail. They have different infrared spectra (34,35), different structures (36), and different functions (37), namely catalysis and dioxygen storage. Initially, the origins of the equilibrium fluctuations EF1 (now ␤ h ) and EF2 (now ␣) in Fig.…”

Ligand dynamics in a protein internal cavity

“…This primary photoproduct has been characterized by x-ray cryocrystallography of photolyzed MbCO crystals at 20-40 K (4-6). Fourier transform IR (FTIR) spectroscopy shows two distinct stretch bands for CO in state B (7,8) that originate from two opposite orientations of the CO dipole with respect to an internal electric field. Recent femtosecond IR experiments (8) and time-resolved x-ray structure determinations (9) at room temperature have confirmed the relevance of docking site B in physiological ligand binding.…”