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Binding of carbon monoxide to the beta chain of adult human hemoglobin has been studied by flash photolysis over the time range from about 100 ps to seconds and the temperature range from 40 to 300 K. Below about 180 K, binding occurs directly from the pocket (process I) and is nonexponential in time. Above about 180 K, some carbon monoxide molecules escape from the pocket into the protein matrix. Above about 240 K, escape into the solvent becomes measurable. Process I can be observed up to 300 K. The low-temperature data extrapolate smoothly to 300 K, proving that the results obtained below 180 K provide functionally relevant information. The experiments show again that the binding process even at physiological temperatures is regulated by the final binding step at the heme iron and that measurements at high temperatures are not sufficient to fully understand the association process.

Binding of carbon monoxide to .8 chains of hemoglobin Zurich has been studied by flash photolysis over the time range of nanoseconds to seconds at temperatures from 20 to 300 K. From 20 to 200 K a single rebinding process (process I) is seen, characterized by a distribution of barrier heights with a peak enthalpy of 2.3 kj/mol. Above 200 K some ligands escape from the pocket into the matrix, and above 260 K recombination from the solvent sets in. Process I is visible up to 300 K, but above 200 K its rate remains essentially constant at about 4 X 108 s1. Above about 250 K, process I is exponential in time, indicating rapid conformational relaxation. The results are discussed within the framework of a sequential model for ligand binding.Binding and dissociation of small ligands, such as 02 and CO, to monomeric heme proteins is one of the simplest biological reactions. Early studies near physiological temperatures explained binding as a one-step process in which the ligand, coming from the solvent, overcomes a barrier of unspecified origin to bind at the heme iron (1). The work of Austin et al. (2), using flash photolysis and covering wide ranges in temperature (2-320 K) and time (microseconds to thousands of seconds), led to a model in which a ligand must overcome a series of sequential barriers (2). The M)del implies that proteins exist in a large number of structur lly related conformational substates (2, 3), and that the innermost barrier, at the heme, is still present at physiological temperatures. Moreover, the experiments suggest that even at physiological temperatures the heme barrier, rather than diffusion (4, 5), controls binding (2, 6, 7). Because understanding one simple biological process in full detail may lead to deeper insight into more complex phenomena, it is crucial to prove or disprove the central features of the sequentialbarrier model.In the sequential-barrier model (2, 7, 8), a ligand coming from the solvent (S) migrates through the protein matrix (M) to the heme pocket (B) and overcomes a final barrier at the heme to bind covalently to the heme iron (state A):[1] In a photodissociation experiment, the system starts out in state A. The laser pulse breaks the Fe CO bond and the system moves rapidly from A to B (9). In the heme pocket the ligand is essentially free (10, 11). Further evolution of the system depends on temperature. Below about 160 K, each protein rebinds its ligand from the pocket (process I Aon(T) = kBA(7hPB(T)Nout (7) kBA(T = dHBAg(HBA)kBA(HBAT) [4] [5]where kBA(T) is an average rate coefficient for the step B > A and PB(T) is the probability of finding state B in the limit kBA -* 0.Eq. 5 assumes that the low temperature distribution g(HBA) is also relevant at high temperatures at which it relaxes to its mean value HBA. This point will be discussed below.The central assumptions underlying Eq. 4 are (i) process I still operates at high temperatures, (ii) binding is sequential as 6239 The publication costs of this article were defrayed in part by page charge payment....

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