We have studied the effect of the P6-inositol (IHP)-induced change from the quaternary oxy (R) to the deoxy (T) structure in derivatives of human, trout IV, and carp methemoglobins. Addition of IHP to human fluoroand aquomethemoglobin leads to the appearance of the slowly exchanging proton resonance at about -10 ppm from HDO diagnostic of the T structure. This experiment, and the crystallization of aquomethemoglobin + IHP by G. Fermi & M. F. Perutz ((1977) J. Mol. Biol. 114, 421) confirmed that the spectral change in the UV which IHP induces in these compounds can be used as a reliable indicator of the R-*-T transition. Judged by this spectral change, IHP converts all derivatives of carp hemoglobin from the R to the T structure. The pH at which the midpoint of the IHP-induced transition occurs increases with rising spin, being lowest in cyano, intermediate in azido, and highest in thiocyanate and aquomethemoglobin of carp. Conversely the replacement of water by fluoride or thiocyanate as the sixth ligand is unaffected by IHP because all three derivatives are predominantly high spin, but the affinity of azide for carp aquomethemoglobin is reduced 2.7-fold and that of cyanide 3.3-fold by IHP, corresponding to changes in the free energy of binding of 600 and 700 cal/mol heme. Conversion to the T structure of all carp methemoglobin derivatives except the cyanide one is accompanied by large changes in the visible absorption spectra, the most spectacular being that of the nitrite derivative whose color is changed from red to brown. IHP converts all human methemoglobin derivtleme-heme interaction arises from an equilibrium between states which differ in the tertiary structure of the a and 0 subunits and in their quaternary structure in the tetramer. This equilibrium is linked to the stereochemistry at the heme. In deoxyhemoglobin where the tense (T) quaternary structure is dominant, the heme irons are five coordinated and high spin.

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The kinetics of the interaction between deionized supernatant aspartic aminotransferase and various anions (cacodylate, phosphate and chloride) were studied by the temperature-jump technique.The anion concentration in the range covered by our experiments does not affect the transamination rate. On the other hand the conformational transition, recently observed at the active site of the enzyme, is hindered by an excess of anions.A single relaxation effect was observed at the enzyme chromophore wavelength in systems containing the aldimine form of the enzyme and the above anions. It is shown that this effect corresponds to the protonation of the chromophore. The relaxation times were of about 10 ps with phosphate, 20-100 ps with cacodylate and 1 -2 ms with chloride. The pH and concentration dependence of this effect were studied. The fits of experimental data to a rate equations for various models were tested by a x2 analysis. The best fit was obtained with models where anions bind rapidly to a site close to the chromophore, so that the pK of the chromophore is affected by anions binding. The rate of the observed relaxation considerably increased when the anion has buffering capacities ; this indicates, in the case of cacodylate and phosphate, that the acidic component of the buffer directly exchanges a proton with the enzyme chromophore.The role which substrate binding, per se, may play in bringing about the large and specific rate acceleration typical of enzyme-catalyzed reactions has recently been the focus of considerable atten-One interesting aspect of the binding of charged substrates to enzymes is the interaction with ionic components of the reaction medium. A pH-dependent competition between anions and substrates or analogs has been reported for soluble aspartate aminotransferase [2]. Such competition could exert an important regulatory role [3] and should be included in overall reaction mechanisms for this enzyme.The equilibrium constants for the binding of protons, various anions, substrates and dicarboxylic substrate analogs to the aldiminic and aminic forms of aspartate aminotransferase have recently been measured by spectrophotometric and nuclear magnetic resonance titrations [2-111.Previous kinetic studies were focused on the interaction of the enzyme with substrates and analogs [12,13] and a fast pH-dependent conformational transition involving the active site of aspartate aminotransferase [14] has recently been reported by this laboratory. Very little information exists about the Enzyme. Aspartate aminotransferase (EC 2.6.1 .I) rates of the interaction between the enzyme and the anions from the medium.It is the object of the present paper to describe some kinetic aspects of this interaction : the influence of anion binding both on the conformational flexibility and on the proton affinity of the chromophore were studied, and the results were correlated with previous findings and with current views on the detailed mechanism of this enzyme. MATERIALS AND METHODSCytoplasmic L-aspartate aminotransfer...

system CuC1,-LiC1-acetonitrile, where CuC142-predominates in the equilibrium mixture, leads to formation of a transient which corresponds to the C1, -radical anion and is not scavenged by the solvent. COPPER(II) CHLORIDE dissolved in acetonitrile gives rise to an equilibrium mixture of tetraco-ordinated CuII species in g, 5