title: Time-resolved absorption and UV resonance Raman spectra reveal stepwise formation of T quaternary contacts in the allosteric pathway of hemoglobin.
abstract: 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 micros and 21 micros, instead of a single 20 micros process. The first step involves the "hinge" region contacts, as monitored by the Trp beta 37...Asp alpha 94 H-bond, while the second involves the "switch" region, as monitored by the Tyr alpha 42...Asp beta 99 H-bond. A working model for the allosteric pathway is presented.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
authors.forename: Gurusamy
authors.initials: G
authors.lastname: Balakrishnan
authors.forename: Martin A
authors.initials: MA
authors.lastname: Case
authors.forename: Alex
authors.initials: A
authors.lastname: Pevsner
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Zhao
authors.forename: Charbel
authors.initials: C
authors.lastname: Tengroth
authors.forename: George L
authors.initials: GL
authors.lastname: McLendon
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Spiro
mesh_terms: D000042:Absorption; D000494:Allosteric Regulation; D001216:Asparagine / Q000737:chemistry; D002263:Carboxyhemoglobin / Q000737:chemistry; D019281:Dimerization; D006441:Hemoglobin A / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010782:Photolysis; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012015:Reference Standards; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D014443:Tyrosine; D001216:Asparagine; D014364:Tryptophan; D006441:Hemoglobin A; D002263:Carboxyhemoglobin