Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains.

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.

Kinetics of p-mercuribenzoate binding to sulfhydryl groups on the isolated cytoplasmic fragment of band 3 protein. Effect of hemoglobin binding on the conformation.

Hemoglobin binds to the cytoplasmic domain of band 3 protein (CDB3) at physiologic pH and ionic strength in an oxygen-linked fashion, with deoxyhemoglobin having the higher affinity. The evidence in the literature suggests functional communication between the hemoglobin-binding site on CDB3 and the anion transport sites within the membrane-bound domain of band 3. Since the hemoglobin-binding site is estimated to be over 200 A from the transport domain, the functional communication hypothesis would require the existence of long-range, global changes in the CDB3 dimeric quaternary structure consequent to hemoglobin binding. In this report sulfhydryl reactivity toward p-mercuribenzoate is studied in an attempt to identify such long-range conformational changes. Formation of stoichiometric hemoglobin/CDB3 complexes is shown to produce major changes in sulfhydryl reactivity. Since the sulfhydryl pocket of CDB3 is known to lie at the dimeric interface over 100 A from the hemoglobin-binding site, the observed changes in reactivity suggest that hemoglobin complexation induces a global change in quaternary structure of the CDB3 dimer. This change offers a mechanism to explain functional connections between CDB3-binding sites and the anion transport sites on band 3. The existence of such long-range conformational changes would imply that the CDB3 dimer is poised to function as a cytosolic arm or lever in order to modulate the global structure of the porter.

Preparation and properties of nickel hemoglobin.

Hemoglobin A reconstituted with nickel protoporphyrin IX (NiHbA) has been prepared and characterized. Kinetics of its reaction with p-mercuribenzoate and with haptoglobin, absorption and circular dichroism spectra, and x-ray crystallographic properties have been investigated as probes of its structural conformation. The results suggest that NiHbA exists in a structure that is similar to the deoxy, or T-state of HbA. It is proposed that NiHbA and its derivatives may serve as a useful model for future studies of hemoglobin allosteric changes.

Catalytic effects by thioltransferase on the transfer of methylmercury and p-mercuribenzoate from macromolecules to low molecular weight thiol compounds.

Thiol agarose and glyceraldehyde-3-phosphate dehydrogenase were blocked with methylmercury or p-mercuribenzoate. The exchange of mercurials between the thiol-containing polymers and glutathione or dithioerythritol was investigated. The activity of glyceraldehyde-3-phosphate dehydrogenase was inhibited by blocking thiol-groups with the mercury compounds. Inhibition was reversible when a short period of inactivation was used. Inactivation for longer periods resulted in reduced regain of enzyme activity. The activity was in part regained when either of the 2 thiol compounds was added. Thioltransferase, known to catalyze thiol-disulfide exchange reactions, increased the regain of glyceraldehyde-3-phosphate dehydrogenase activity to nearly the original value. Here, thioltransferase is proposed to catalyze the transfer of organomercurial from one thiol complex to another. Some consequences of the observations in vivo are discussed.

Homoserine dehydrogenase: spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme--p-mercuribenzoate complex.

Incubation of Rhodospirillum rubrum homoserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of 0.2 M KCl and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCl, and L-threonine, a time-dependent recovery of enzyme activity was observed in 25 mM phosphate/I mM EDTA buffer, pH 7.5. Circular dichroism studies indicated that the transition from inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with [14C]PMB revealed loss of enzyme-bound radioactivity during reactivation. Increase in ionic strength of the phosphate buffer and/or addition of L-threonine, leading to enzyme aggregation, decreased the rate of enzyme reactivation, aggregated enzyme that remained inactive retained [14C]PMB on the enzyme. Sulfhydryl titration of various forms of the enzyme suggested a preferential release of PMB from a sulfhydryl group essential to enzymic activity. We conclude that reactivation of the inactive enzyme is due to dissociation of PMB from an "active-site" sulfhydryl group and that changes in the protein structure influence the rate of dissociation of the enzyme-PMB complex.