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

Abstract

A nanosecond absorption spectrometer has been used to measure the optical spectra of hemoglobin between 3 ns and 100 ms after photolysis of the CO complex. The data from a single experiment comprise a surface, defined by the time-ordered set of 50-100 spectra. Singular value decomposition is used to represent the observed spectra in terms of a minimal set of basis spectra and the time course of their amplitudes. Both CO rebinding and conformational changes are found to be multiphasic. Prior to the quaternary structural change, two relaxations are observed that are assigned to geminate recombination followed by a tertiary structural change. These relaxations are interpreted in terms of a kinetic model that points out their potential role in kinetic cooperativity. The rapid escape of CO from the heme pocket compared with the rate of rebinding observed for both R and T quaternary states shows that the quaternary structure controls the overall dissociation rate by changing the rate at which the Fe-CO bond is broken. A comparable description of the control of the overall association rates must await a more complete experimental description of the kinetics of the quaternary T state.Beginning with the work of Gibson (1), experiments on the photolysis of the CO complex of Hb have played a central role in investigations of the mechanism of cooperative ligand binding (2). Hopfield et al. (3) proposed that the major features of the bimolecular recombination kinetics could be rationalized by a two-state allosteric model in which the R and T quaternary structures are in equilibrium. Subsequent studies have focused on measuring the rate of the quaternary structural transition (4-6). Recent improvements in time resolution have introduced new dimensions to the binding mechanism. Transient absorption spectroscopy using 10-ps laser pulses at room temperature showed no evidence of CO rebinding in <1 ns (7,8). Significant recombination of CO was observed, however, at 50-100 ns in experiments using 10-to 30-ns laser pulses (9-13). 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. Our approach has been to measure precise optical absorption spectra over a wide range of time (from ""3 ns to 100 ms) following photolysis of HbCO. The results suggest a kinetic model that gives additional insight into the nature of the elementary processes responsible for cooperativity. MATERIALS AND METHODSHuman HbO2 A was purified by column chroma...

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

“…Unliganded crystals of human hemoglobin grown under certain low-salt conditions can maintain their integrity upon oxygen binding; however, oxygen binding in this case is noncooperative (12). Although the ability to follow allosteric transitions in the crystalline state is limited when such large quaternary structural changes accompany cooperative ligand binding, spectroscopic investigations of hemoglobin solutions have revealed some aspects of the kinetic pathway of allosteric structural transitions (13)(14)(15). Results to date suggest a multistep pathway, with key tertiary structural transitions taking place within a few microseconds and quaternary rearrangements occurring in the range of tens of microseconds.…”

Conformational diversity of acid‐denatured cytochrome c studied by a matrix analysis of far‐UV CD spectra

“…For that purpose, we have applied a singular value decomposition (SVD) analysis on a large set of the far-UV CD spectra of cyt c (typically more than 200 spectra). Application of SVD to spectroscopic or other data sets of proteins is not at all new, and is even classical (Hofrichter et al, 1983;Henry & Hofrichter, 1992;McPhie & Shrager, 1992;Jones et al, 1993;Ansari et ai., 1994;Sreerama & Woody, 1994;Chen et al, 1996;Jager et al, 1997). Here, we use it to analyze far-UV CD spectral changes produced by co-solvents.…”

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin

“…Following laser photodissociation of CO, the early (147 ns) post-photolysis signal evolved toward the well defined deoxyHb vs. HbCO scattering difference pattern in about 2 μs. This estimate implies that dimer rotationthe largest structural change in the R-T allosteric transition-is much faster than 20 μs, the time constant detected by optical spectroscopy (2,3). Quite recently Fischer et al (14) published a computational analysis of Hb dynamics which revealed distinct tertiary and quaternary conformational events; in particular, they reported two sequential quaternary changes (called Q 1 and Q 2 ), which they assumed to be consistent with the time constants at 2 μs (for Q 2 ) and 20 μs (for Q 1 ) reported by Balakrishnan et al (10).…”

“…Starting with the carbon monoxide adduct HbCO in the allosteric quaternary state called R 4 , complete photolysis yields the unliganded R 0 state; the destiny of this photoproduct is a complex time-dependent process involving competing events such as ligand rebinding and (tertiary and quaternary) conformational decays. Changes in the optical and resonance Raman spectra of the different states have provided, over the last four decades, a quantitative estimate of the rates of the competing events (2)(3)(4)(5). For a review on time-resolved optical absorption (TR-OA) data describing conformational decays as well as rebinding in the dark of a ligand escaped into the solvent (bimolecular) or trapped within the protein matrix (geminate), see Eaton et al (6).…”

“…The much larger structural changes of the quaternary allosteric transition (involving shifts in the inter-subunit contacts and relative motions of the two symmetric dimers α 1 β 1 ∕α 2 β 2 ) occur in the microsecond time range; the single time constant estimated from optical changes of the deoxy photoproduct is about 20 μs for human HbA at neutral pH and 20°C (2,3,6,7). The dynamics of the structural changes followed by ultraviolet resonance Raman (UVRR) spectroscopy after laser photolysis showed, however, the quaternary conformational transition involving contacts at the α 1 β 2 and α 2 β 1 interfaces (8, 9) to be more complex.…”