Transient haem–globin interactions in photodeligated carboxyhaemoglobin and subunits

Abstract

Although the fixation of ligand to haemoglobin (Hb) is known to be accompanied by changes in protein conformation regulating the oxygen exchange in blood, the mechanism triggering these changes remains undecided. We now report a dynamic approach to this problem using results obtained in a nanosecond laser photolysis study of carboxyhaemoglobin (HbCO) and its isolated subunits. The study is based on our previous observation of a structural evolution of free Hb after photodeligation, manifested through slight variations of the protein spectrum in the microsecond range. It is now found that the isolated subunits also show this behaviour. The duration of the spectral evolution is approximately 2 microseconds for the three proteins and the activation energy of the process approximately 9 kcal mol-1. The spectral evolution is attributed to local conformation changes at the haem region, occurring during the structural relaxation of the freshly deliganded protein. The results for the isolated chains show that such changes exist even in the absence of the R-T transition.

Nanosecond absorption spectroscopy of hemoglobin: elementary processes in kinetic cooperativity.

“…Because the same spectral change is observed with the same rate and amplitude in the partial photolysis experiment (c) ( Table 1), in which there is both kinetic and spectroscopic evidence that there is no R -* T transition, the associated structural change can be considered as a pure tertiary change. A tertiary change in this time regime has been previously proposed on the basis of transient Raman (14) and single-wavelength absorption studies (15). There is a small change in fractional saturation associated-with relaxation II, suggesting geminate rebinding from a second nonheme binding site in the globin (13).…”

“…This has been interpreted as the rebinding of photodissociated CO that has not yet escaped from the protein into the solvent and is called "geminate" recombination (9-13). At later times, both optical absorption and resonance Raman spectra of the deoxy hemes showed spectral changes in 0.5-5 ,us, which have been interpreted as resulting from tertiary conformational changes of the globin (14,15).No kinetic model has been formulated that relates geminate rebinding to heme stereochemical and globin conformational changes or to the overall thermal ligand dissociation and binding reactions. A major objective of the present study is to make this connection.…”

“…This has been interpreted as the rebinding of photodissociated CO that has not yet escaped from the protein into the solvent and is called "geminate" recombination (9)(10)(11)(12)(13). At later times, both optical absorption and resonance Raman spectra of the deoxy hemes showed spectral changes in 0.5-5 ,us, which have been interpreted as resulting from tertiary conformational changes of the globin (14,15).…”

Metastable Species of Hemoglobin: Room Temperature Transients and Cryogenically Trapped Intermediates

“…Time-resolved studies of transients have been limited to optical absorption (11)(12)(13)(14) and resonance Raman spectroscopy (1) (5,6), and infrared absorption spectroscopy (4). Optical absorption studies (4) of lowtemperature metastable species have not revealed differences between deoxy species and photolyzed species owing to incomplete sample photolysis.…”

Time‐resolved resonance Raman studies of carp hemoglobin

“…On this time scale, the heme has relaxed to an Fe z+ high-spin deoxy heme with the iron displaced from the plane of the porphyrin [2,3]. Large scale motions of protein that influence the heme environment are, however, not detectable until many 100's of ns later [3,14,15]. Consequently we have at 10 ns, a deoxy-heme surrounded by the heme environment that 10 ns earlier had been associated with the ligand-saturated porphyrin and is now, by virtue of the induced ligand dissociation, rendered unstable.…”