Time-resolved Absorption and UV Resonance Raman Spectra Reveal Stepwise Formation of T Quaternary Contacts in the Allosteric Pathway of Hemoglobin

Hemoglobin undergoes a series of molecular changes on the nanosecond and microsecond time-scale following photodissociation of CO ligands. We have monitored these processes with a combination of transient absorption and resonance Raman (RR) spectroscopy. The latter have been acquired at higher data rates than previously available, thanks to kilohertz Ti : sapphire laser technology, with frequency-quadrupling into the ultraviolet. As a result of improved resolution of the UVRR time-course, a new intermediate has been identified in the pathway from the R (HbCO) to the T (deoxyHb) state. This intermediate is not detected via absorption transients, since the change in heme absorption is insignificant, but its lifetime agrees with a reported magnetic circular dichroism transient, which has been attributed to a quaternary tryptophan interaction. The new UVRR data allow elaboration of the allosteric pathway by establishing that the T-state quaternary contacts are formed in two well-separated steps, with time constants of 2.9 μs and 21 μs, instead of a single 20 μs process. The first step involves the “hinge” region contacts, as monitored by the Trpβ37⋯Aspα94 H-bond, while the second involves the “switch” region, as monitored by the Tyrα42⋯Aspβ99 H-bond. A working model for the allosteric pathway is presented.