Nanosecond flash photolysis study of carbon monoxide binding to the beta chain of hemoglobin Zürich [beta 63(E7)His leads to Arg].

Abstract

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....

Perturbations of the distal heme pocket in human myoglobin mutants probed by infrared spectroscopy of bound CO: correlation with ligand binding kinetics.

“…The rebinding barrier height for SW H64G below 160 K was found to be smaller than for native SW MbCO, which is in agreement with our results for human Mb H64A. Recently, a study of Leu-29 mutants in human Mb showed an increase in a low-frequency IR peak, and the bimolecular CO rebinding of these mutants was found to be (40,41), consistent with the trends we are seeing.…”

Conformational substates in azurin.

“…3a) is a phenomenon that is familiar to us from studies of heme proteins in this temperature range (1,2,25,26). In heme proteins, the nonexponential behavior has been explained with the concept of CS: At low temperatures, the proteins are frozen into many slightly different structures, the CS, with different activation enthalpies, HBA, for ligand binding.…”

“…In heme proteins, the nonexponential behavior has been explained with the concept of CS: At low temperatures, the proteins are frozen into many slightly different structures, the CS, with different activation enthalpies, HBA, for ligand binding. The inhomogeneous ensemble of protein molecules is characterized by a distribution of enthalpy barriers, g(HBA).t If we assume that kAB << kBA, the fraction of proteins that have not yet rebound a ligand at time t after photodissociation is described by N(t, T) = f g(HBA)e-kBA(HBT)tdHBA [31 The temperature dependence of the rate coefficient kBA(HBA, T) is usually given by the transition-state expression (26) kBA(HBA, T) = ABA(T/To)eHBAIRT.…”

Recombinant [Pheβ63]Hemoglobin Shows Rapid Oxidation of the β Chains and Low‐Affinity, Non‐Cooperative Oxygen Binding to the α Subunits

“…The influence of the distal His (E7) residue on the functional properties of Mb and Hb has been studied for natural and recombinant mutants. For example, the natural Hb Zurich ([Argp63] Hb) exhibits a substantial increase in the rate of CO recombination after photodissociation (Dlott et al, 1983;Scott et al, 1985). This increase has been attributed to the effect of the large guanidinium group of arginine, which exchanges hydrogen bonds with the propionate side chains of the heme and contributes to enlarge the entrance of the heme pocket (Tucker et al, 1978).…”