Geminate recombination of O2 and hemoglobin.

Abstract

The photolysis of HbO2 and HbCO has been studied by measuring transient absorption spectra in the Soret region after excitation with picosecond pulses at 530 nm. Dissociation occurred promptly in both cases, followed (for HbO2) by geminate recombination of ca. 40% of the photodissociated O2 with a lifetime of 200 ± 70 psec (25°C). No to 300 psec. The initial MbCO photoproduct signal at 440 nm was reported to undergo a 15% decay (lifetime r = 125 + 50 psec), but for MbO2 no evidence of decay up to 450 psec was found. Greene et al. (8) conducted a study of HbO2 and HbCO photolysis using 8-psec, 353-nm excitation. They obtained transient absorption spectra in the Soret (400-450 nm) and visible (535-575 nm) regions at 10 psec and 680 psec, finding a persistent, broadened deoxy Hb-like absorption. With their spectra, they observed less than 10% recombination in HbCO over this time interval and less than 20% recombination of photolyzed HbO2. Greene et al. (8) found that HbCO responded similarly to 530-and 358-nm pulses. The present study of the 530-nm photolysis of HbCO and HbO2 is aimed at identifying the species initially generated by light and how these species evolve.Photodissociation of liganded hemoglobins is a well-known phenomenon (1) used to study dynamics of ligand binding to deoxyhemoglobin (Hb) (2)(3)(4)(5)(6)(7)(8). The usefulness of the photoprocess for subsequent dynamical studies is restricted at present by a lack of knowledge of the mechanism of the photodissociation. Of particular interest is the quantum yield for dissociation under steady illumination, which is fairly high (k 0.5) (9, 10) for carboxyhemoglobin (HbCO) but much lower (0 0.05) for the oxy form (HbO2) (9). Two limiting mechanisms can account for the relatively low yield of HbO2 photolysis. First, energy relaxation to nonreactive states may be much faster than the dissociation. Second, although the initial dissociation step in HbO2 may be just as efficient as that in HbCO, some released 02 trapped nearby to the heme may be able to rapidly recombine. These mechanisms are evaluated here by examining the dynamics and identifying the products of the photolysis by picosecond spectroscopy. We have attempted to describe our spectroscopic and dynamical results in terms of recent experimental and theoretical assessments of the excited states of hemoglobins (11-15).The time scale for studying photolysis and subsequent events in heme proteins has in recent years been pressed back to the picosecond regime, starting with the work of Shank et al. (5). Using subpicosecond pulses at 615 nm to excite and probe HbO2 and HbCO, they found induced absorption build-up in less than 0.5 psec for both species, a decay of the HbO2 signal in 2.5 psec, and no decay of the HbCO signal up to 20 psec. Rentzepis and coworkers (6, 7) studied HbCO and the myoglobin compounds MbCO and MbO2 by exciting with 6-psec 530-nm pulses and using 440-nm interrogation over the period 0-00 psec. That group reported a significantly longer (11 psec) predissociation lifetime...

Picosecond fluorescence decay of tryptophans in myoglobin.

“…We have therefore carried out picosecond laser experiments on Mb aimed at characterizing the fluorescent behavior of each of the tryptophans. Previous picosecond laser experiments aimed at detecting rapid conformational changes and relaxation processes in heme proteins have involved studying changes in the heme electronic structure as a result of photodeligation of CO (1)(2)(3)(4)(5)(6), 02 (1,(4)(5)(6), and NO (6,7). A goal of the present research is to pave the way for the study of the transmission of structural changes throughout the protein, and as a first step we have attempted in the present work to chart the picosecond anatomy of the tryptophans of Mb.…”

On a Research Rollercoaster With Friends

“…The dissociation of CO was immediate and there was no geminate recombination. The dissociation of oxygen from HbO 2 was also immediate but we discovered a 200-ps geminate recombination of the oxygen molecules (61). The nitric oxide ligand of HbNO dissociated immediately followed by a very rapid nonexponential geminate recombination of the NO (62).…”

Time‐resolved resonance Raman studies of carp hemoglobin

“…One such process that is readily studied is geminate recombination [7][8][9] which is known to occur both on the sub-ns [16] and many 10's of ns [7][8][9] time scale for photodissociated 02. Recent studies (in preparation) on oxy-and nitroxy-Hb's have revealed that for human Hb and turtle Hb the yield of geminate recombination occurring over a 10 ns pulse correlates with the frequency of both v4 and the Fe-His stretching mode which are themselves correlated.…”