Subunit-selective interrogation of CO recombination in carbonmonoxy hemoglobin by isotope-edited time-resolved resonance Raman spectroscopy

Abstract

Hemoglobin (Hb) is an allosteric tetrameric protein made up of alphabeta heterodimers. The alpha and beta chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with (15)N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with chain selectivity by resonance Raman (RR) spectroscopy. For alpha but not for beta chains, the frequency of the nu(4) porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation of proximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the alpha chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 +/- 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the alphabeta interface, evidence for which has emerged from UVRR studies of site mutants.

Figure 1

Static RR spectra excited at 426 nm (0.3 mW) for the deoxy and CO-adducts of HbA and of the (¹⁴α ¹⁵β)₂ isotope hybrid.

Figure 2

Time-resolved RR spectra at the indicated time (µs) following photolysis of the CO adduct of HbA and of the (¹⁵α ¹⁴β)₂ and (¹⁴α ¹⁵β)₂ hybrids. Band positions and assignments are marked at the top. Samples were prepared at a heme concentration of ~0.25 mM in CO saturated 50 mM sodium phosphate buffer. [λ_pump = 419 nm, 65 mW, ~20 ns, 1 kHz; λ_probe = 426 nm, 0.5 mW, ~20 ns, 1 kHz].

Figure 3

The CO rebinding to photolyzed CO adducts of HbA and the indicated Hb hybrids, determined from the intensity of the ν₄ CO-heme band. The continuous lines correspond to modeling of the CO recombination reaction with the time constants(40), τ_gem (geminate phase), τ₄ (R-state bimolecular rebinding), and τ₅ (T-state bimolecular rebinding).

Figure 4

Example of RR band deconvolution at 3, 50 and 300 µs following CO photolysis, showing the experimental data (black) fitted spectra (green), deconvoluted deoxy bands (red), CO bands (blue) and the residuals (gray).

Figure 5

Deconvolution data showing that ν₄ shift with time only for α- subunits in the deoxy state.

Figure 6

Time evolution (log scale) for the deoxy ν₄ frequencies of HbA and for the α- subunits in (¹⁵α ¹⁴β)₂ and (¹⁴α ¹⁵β )₂ hybrids, following CO-photolysis. The continuous lines correspond to modeling with a series of time constants (τ₁= 65 ns; τ₂’= 0.74 µs_; τ₂= 2.9 µs_; τ₃ = 20.5 µs_; τ_4,5 = 497 µs ) previously documented for protein structural changes from UVRR spectroscopy(34).

Figure 7

Diagram of the EF clamshell rotation proposed(34, 36) to follow HbCO photodissociation. Displacement of the high-spin Fe from the heme plane impels the F helix away from the heme, while E helix movement toward the heme expels CO from the heme pocket or else induces geminate recombination.