Carboxy Mb at pH 3. Time-resolved resonance Raman study at cryogenic temperatures

Abstract

Cryogenic samples of MbCO at pH3 are studied using nanosecond and picosecond time-resolved resonance Raman spectroscopy. It is observed that under excitation conditions sufficient to completely photodissociate MbCO at pH7, the pH3 sample at 10 ns remains substantially unphotolyzed even at 15 K. The similarity in the optical and resonance Raman spectra of MbCO at pH3 with that of pH7 indicates that at pH3 the iron remains six-coordinate and low-spin. The Fe-CO stretch frequency is consistent with a more upright CO orientation. The absence of the v(Fe-His) band in the 30 ps photoproduct Raman spectrum suggests that the Fe-His(F8) bond is broken within 30 ps of photodissociation. Other Raman bands, though, are not consistent with a normal four-coordinate heme for the photoproduct, Mb*. Suggested possible interpretations include a four-coordinate heme highly perturbed by the close lying protonated proximal histidine or a five-coordinate heme with the Fe-His bond significantly weakened. The partial photolysis monitored at 30 ps and 100 K indicates either a significant amount of geminate recombination within 30 ps or low quantum yield or photolysis. The time course for CO recombination is monitored via the Raman spectra from 30 ps to 3 ns at 100 K and 160 K. Of the fraction of protein-ligand pairs that remain photodissociated at 30 ps, 50% recombine by approximately 250 ps at 100 K and 160 K, supporting the flash photolysis rebinding data of Cowen et al. (Cowen, B. R. 1990. Ph. D. thesis. University of Illinois at Urbana-Champaign; Cowen, B. R., D. Braunstein, H. Frauenfelder, P. J. Steinbach, and R. D. Young. 1989. Biophys. J. 55:55a. [Abstr.].) The conclusions from these resonance Raman studies are extended to solution phase studies at ambient temperatures.

Low pH myoglobin photoproducts

“…The purpose of this paper is to try to reconcile these apparently contradictory observations. We point out some inconsistencies in a previous attempt (9) to explain both high and low temperature observations and suggest a simple modification of the scheme originally proposed to describe the room temperature measurements (7) that is also consistent with the low temperature observations (see Scheme 1).…”

“…To our knowledge, the existence of either a doubly-protonated histidine nitrogen or a high spin four-coordinate heme is unprecedented, and this scheme must be regarded as speculative. Nevertheless, it does provide a consistent description of the low temperature measurements (9). Iben et al (9) also make an ingenious attempt to describe the room temperature results within the same scheme.…”

“…In principle, flash photolysis is a preferable method for studying ligand rebinding to four-coordinate hemes, because it should allow observation of the geminate phase. However, although MbCO is partially unfolded below pH4 (8) (also see below), the iron-histidine bond remains intact (6,7,8,9), and rebinding to a fourcoordinate heme can only be observed if the proximal bond ruptures before CO rebinding. Two recent studies (7, 9) have addressed this issue but arrived at apparently contradictory conclusions: at room temperature, the 220-cm-1 mode associated with iron-histidine stretching is clearly present in the 10-ns photoproduct spectrum (7), while the absence of this mode in the spectrum of photolyzed MbCO in a glycerol/water glass at 100-K has been interpreted as evidence for rupture (or weakening) of the iron-histidine bond within 30 ps of photolysis (9).…”

“…This scheme invokes an unusual situation in which histidine remains bound to the iron in MbCO, but the b-nitrogen of the imidazole becomes doubly protonated at low pH (species 2'A). Upon photolysis, Iben et al (9) suggest that either (a) the iron-histidine bond breaks, but the proximity of the charged histidine pushed the iron out of the heme plane toward the distal side and the iron remains high spin or (b) the bond remains intact, but the presence of the extra proton on N8 leads to a drastic change in either the ----H20…”

Kinetic Modulation in Carbonmonoxy Derivatives of Truncated Hemoglobins

“…Picosecond CO recombination has been observed for COMb at low pH under cryogenic conditions where the iron-proximal histidine bond is broken (57). By eliminating the energetic cost of moving the iron into the heme plane upon ligand binding, the loss of the iron-proximal histidine bond results in a very substantial decrease in Barrier I.…”