Active sites isolation, discussion

In this section we review the important theoretical developments relevant to host-sensitized energy transfer. The basic interaction between two isolated ions or molecules is discussed first, then the effects of having an ensemble of sensitizer and activator ions is presented. The mathematical description of energy transfer by multi-step migration among sensitizer ions is developed and the effects of trapping at activator sites is discussed. The importance of phonons in both single-step and multi-step transfer processes is also described. 

The subunits of CODH/ACS have been isolated (see earlier discussion). The isolated a subunit contains one Ni and four Fe and has spectroscopic properties (186) similar to those of Cluster A, the active site of acetyl-CoA synthesis (212). Unfortunately, it has no ACS activity. Therefore, ACS activity may reside in the a subunit or it may require both the a and the fi subunits. If Clusters B and/or C of the B subunit are involved in acetyl-CoA synthesis, one possible role could be in electron transfer. Although acetyl-CoA synthesis and the CO/ exchange reactions do not involve net electron transfer, both of these reactions are stimulated by ferredoxin, indicating that internal electron transfer within CODH/ACS may be required during the reaction (121). Further studies with the isolated subunits and the reconstitu-... 

In 1996 Stack and co-workers reported an unusual 3 1 (copper 02 stoichiometry) reaction between a mononuclear copper(I) complex of a A-permethylated (lR,2R)-cyclohexanediamine ligand with dioxygen. The end product of this reaction, stable at only low temperatures (X-ray structure at —40 °C) is a discrete, mixed-valence trinuclear copper cluster (1), with two Cu11 and a Cu111 center (Cu-Cu 2.641 and 2.704 A).27 Its spectroscopic and magnetic behavior were also investigated in detail. The relevance of this synthetic complex to the reduction of 02 at the trinuclear active sites of multicopper oxidases4-8 was discussed. Once formed, it exhibits moderate thermal stability, decomposed by a non-first-order process in about 3h at —10 °C. In the presence of trace water, the major isolated product was the bis(/i-hydroxo)dicopper(II) dimer (2). 

Porphyrins and their metal complexes, respectively, occur as active sites in a number of enzymes. A-confiised porphyrins such as 75 have been discussed regarding their analogy to NHCs and their potential importance in porphyrin chemistry. Isolation of the first metal complexes containing palladium. 

Purple acid phosphatase (PAP) or tartrate-resistant phosphatase is not thought to be a protein phosphatase but it has a very similar dimetallic active site structure to that found in protein phosphatases. PAPs have been identified in bacteria, plants, mammals, and fungi. The molecular weights (animal 35 kDa, plant 55 kDa) are different and they exhibit low sequence homology between kingdoms but the residues involved in coordination of the metal ions are invariant. " There has been considerable debate as to the identity of the metal ions in PAPs in vivo. Sweet potato, Ipomoea batatas, has been shown to possess two different PAP enzymes and the active site of one of them has been shown to contain one Fe and one Zn " " ion. Another report has established that the active site of a PAP from sweet potato contains one Fe " and one Mn +. The well-characterized red kidney bean enzyme and the soybean enzyme contain Fe " and Zn. Claims that PAP from sweet potato has 2Fe ions or 2Mn ions have been discussed elsewhere. One explanation is that these are different forms of the enzyme, another is that because the metal ions are labile and are rapidly incorporated into the active site, the enzyme contains a mixture of metal ions in vivo and the form isolated depends on the conditions of isolation. 

Furthermore, physical processes such as internal and external rotation of intermediates will be indispensable elementary processes in some catalytic reactions. In this review, the discussions are focused on rather simple reactions taking place on isolated single sites, but it may be reasonable to assume that more complex reactions demand more complicated prerequisites for active sites. 

As discussed earlier, the enzymic reaction catalyzed by glutamine synthetase requires the presence of divalent metal ions. Extensive work has been conducted on the binding of Mn2+ to the enzyme isolated from E. coli (82, 109-112). Three types of sites, each with different affinities for Mn2+, exist per dodecamer n, (12 sites, 1 per subunit) of high affinity, responsible for inducing a change from a relaxed metal ion free protein to a conformationally tightened catalytically active protein n2 (12 sites) of moderate affinity, involved in active site activation via a metal-ATP complex and n3 (48 sites) of low affinity unnecessary for catalysis, but perhaps involved in overall enzyme stability. The state of adenylylation and pH value alter the metal ion specificity and affinities. 

In the presence of PPi, known to bind strongly to the enzyme active site (Section III,E), there was a weak protective effect. The experimental points fell in the shaded area of Fig. 6, and the data were analyzed with equations developed by Scrutton and Utter (45). The results of this treatment led to the conclusion that TNBS can react with both free enzyme and enzyme-PPi complex to cause catalytic inactivation the differences are only quantitative (45). Either TNBS can displace PP, from an active site lysine or TNBS modifies a different lysine, apart from the active site, and the presence of PPi on the enzyme partially protects against TNBS inactivation by some indirect mechanism. Unfortunately, as discussed above, this issue cannot be settled with these kinetic analyses. Furthermore, because all of the enzyme lysines are to some extent reactive with TNBS (Fig. 5), the single super-reactive lysine whose modification leads to inactivation cannot be isolated and identified, as, for example, in a particular peptide fragment. A variety of interpretations are possible, as discussed elsewhere (45). 

The metal coordination environment for HPP dioxygenase appears to be an exception to the 2-His-l-Asp active site discussed. This enzyme is isolated in the high-spin Fe(III) state as indicated by an intense EPR signal at g = 4.3. More significantly, it is deep blue in color (A,max 595 nm) and exhibits a resonance Raman spectrum characteristic of a tyrosinate ligand [219], Indeed, sequence comparisons of seven HPP dioxygenases from various mammalian and pseudomonad sources indicate the presence of a conserved Tyr residue [220,221], It... 

Before one tries to construct a steric picture of the events that occur at the active site of the lyase, it is necessary to discuss the history of the re-isolated substrate, which also became labelled with tritium during the above experiments. In order to locate the tritium in these 2-aminopropanols, they were dissolved in deuterium oxide and incubated with HLADH and diaphorase [33]. Although NMR monitoring... 

---