Spectroscopic probes active site

The microenvironment of the micellar core inferred from fluorescent probes is said to be similar to some organic media (Turner and Brand, 1968 Cordes and Gitler, 1973). Similar conclusions have been obtained by other spectroscopic means (see previous sections). The active site of an enzyme is usually quite hydrophobic and the number of water molecules at the active site is limited. Therefore, it is very useful to study the behavior of the catalytic groups in organic media in relation to micellar and enzymatic catalysis. 

As will be explained in Chapter 7, spectroscopic methods are a powerful way to probe the active sites of the hydrogenases. Often spectroscopic methods are greatly enhanced by judicious enrichment of the active sites with a stable isotope. For example, Mossbauer spectroscopy detects only the isotope Fe, which is present at only 2.2 per cent abundance in natural iron. Hydrogen atoms, which cannot be seen by X-ray diffraction for example, can be studied by EPR and ENDOR spectroscopy, which exploit the hyperfine interactions between the unpaired electron spin and nuclear spins. More detailed information has been derived from hyperfine interactions with nuclei such as Ni and Se, in the active sites. In FTTR spec-... 

Our earlier research on the coupled binuclear copper proteins generated a series of protein derivatives in which the active site was systematically varied and subjected to a variety of spectroscopic probes. These studies developed a Spectroscopically Effective Model for the oxyhemocyanin active slte.(l) The coupled binuclear copper active site in tyrosinase was farther shown to be extremely similar to that of the hemocyanlns with differences in reactivity correlating to active site accessibility, and to the monophenol coordinating directly to the copper(II) of the oxytyroslnase site.(2) These studies have been presented in a number of reviews.(3) In the first part of this chapter, we summarize some of our more recent results related to the unique spectral features of oxyhemocyanin, and use... 

Spectroscopies are also used to experimentally probe transient species along a reaction coordinate, where often the sample has been rapidly freeze quenched to trap intermediates. An important theme in bioinorganic chemistry is that active sites often exhibit unique spectroscopic features, compared to small model complexes with the same metal ion.8 These unusual spectroscopic features reflect novel geometric and electronic structures available to the metal ion in the protein environment. These unique spectral features are low-energy intense absorption bands and unusual spin Hamiltonian parameters. We have shown that these reflect highly covalent sites (i.e., where the metal d-orbitals have significant ligand character) that can activate the metal site for reactivity.9... 

If gadolinium ion, G< 2+, is to be useful as a spectroscopic probe of Ca2+ sites on Ca -ATPase, it must first be demonstrated that Gd2+ can compete with Ca + at these Ca + activator sites,... 

Physicochemical methods, i.e. adsorption of probe molecules followed by varied analytical techniques (gravimetry, chromatography, calorimetry, spectroscopic techniques, etc.) are currently used for estimating more precisely the concentration of the potential active sites.[34 36] However, very few methods are well adapted for this purpose most of the methods employed for the characterization of the acidity of solid catalysts lead to values of the total concentrations of the acid sites (Brpnsted + Lewis) and to relative data on their strength, whereas few of them discriminate between Lewis and Brpnsted acid sites. It is however the case for base adsorption (often pyridine) followed by IR spectroscopy, from which the concentrations of Brpnsted and Lewis sites can be estimated from the absorbance of IR bands specific for adsorbed molecules on Brpnsted or Lewis sites. 

Valuable spectroscopic studies on the dithiolene chelated to Mo in various enzymes have been enhanced by the knowledge of the structure from X-ray diffraction. Plagued by interference of prosthetic groups—heme, flavin, iron-sulfur clusters—the majority of information has been gleaned from the DMSO reductase system. The spectroscopic tools of X-ray absorption spectroscopy (XAS), electronic ultraviolet/visible (UV/vis) spectroscopy, resonance Raman (RR), MCD, and various electron paramagnetic resonance techniques [EPR, electron spin echo envelope modulation (ESEEM), and electron nuclear double resonance (ENDOR)] have been particularly effective probes of the metal site. Of these, only MCD and RR have detected features attributable to the dithiolene unit. Selected results from a variety of studies are presented below, chosen because their focus is the Mo-dithiolene unit and organized according to method rather than to enzyme or type of active site. 

In many cases the Zn2+ ion is known to be tetrahedrally coordinated in the resting enzyme, with one ligand being H20, and that it can be replaced by Co2+, which provides a spectroscopic probe of the active site. The Co2+ ion does not significantly depress the enzymatic activity and sometimes even enhances it. 

A. Metal Substitution as a Spectroscopic Probe for Elucidating Active Site Geometry... 

In vitro, both metals in the active site of SOD can be reversibly removed. This behavior has been elegantly expanded to produce a variety of metal-substituted derivatives. Co, Ni, and Ag derivatives, where the added metal ion may occupy the copper site or the zinc site (and in some cases both), have proven especially valuable in providing new spectroscopic probes of metal-site structure and reactivity in Cu, Zn SOD. Application of in vitro metal substitution to several of the FALS wild-type-like mutants has shown that these variants readily misfold and often exhibit altered metal ion binding. In addition to the in vitro metal studies, recent work has centered on the mechanism of in vivo copper incorporation. CCS is a copper chaperone protein that forms a heterodimer with Zn-loaded SOD to insert copper and activate the enzyme (see Metallochaperones Metal Ion Homeostasis). 

Even for metals typically thought of as spectroscopically silent, the changes in biomolecule chemical shifts upon metal binding provide a probe of the active site. 

Atkins. W M., and Sligar, S G, Tyrosine-96 as a natural spectroscopic probe of the cytochrome P-450cam active site. Biochemistry 29, 1271-1275 (1990). 

Fluorescent Equilibrium Probes. Himel and co-workers (23, 24, 25) have synthesized active-site-directed fluorescent equihbrium probes which are competitive inhibitors of the active site of cholinesterase enzymes. The fluorescence intensity of the probe-enzyme complex is decreased by any foreign molecule (insecticide) which competes with the equilibrium fluorescent probe for the active site of the enzyme or which changes the equilibrium dynamics by exo area reaction with the enzyme. This highly specific and sensitive spectroscopic method is being developed as an analytical method for insecticides (26). 

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