Enzymes spectroscopic studies

Spectroscopic studies have been instrumental in elucidating the catalytic mechanism of Ni-Fe hydrogenases. A great deal of controversy concerning this mechanism arises from the fact that, as the as the X-ray crystallographic analysis has shown, there are at least three potential redox-active species at the enzyme s active site the thiolate ligands (75) and the Fe (65) and Ni (9) ions. 

The [NiFe] hydrogenase from D. gigas has been used as a prototype of the [NiFe] hydrogenases. The enzyme is a heterodimer (62 and 26 kDa subunits) and contains four redox active centers one nickel site, one [3Fe-4S], and two [4Fe-4S] clusters, as proven by electron paramagnetic resonance (EPR) and Mosshauer spectroscopic studies (174). The enzyme has been isolated with different isotopic enrichments [6 Ni (I = I), = Ni (I = 0), Fe (I = 0), and Fe (I = )] and studied after reaction with H and D. Isotopic substitutions are valuable tools for spectroscopic assignments and catalytic studies (165, 166, 175). 

Twenty years ago, the first spectroscopic studies were initiated on hydrogenases, and Mossbauer was first applied in 1982 to characterize the iron clusters present in D. desulfuricans hydrogenase (Kruger et al. 1982). Different enzymes have been studied since through a spectroscopic approach. In the remainder of this section, we will present as an example of these studies the work done in the D. gigas [NiFe] hydrogenase (Huynh et al. 1987 Teixeira et al. 1989). 

Bacillus cereus produces /3-lactamase I and /S-lactamase II. The former does not contain zinc.582 The /0-lactamase II binds two Zn11 per molecule. The use of 270 MHz H NMR spectroscopy shows that resonances attributable to three of the histidyl residues in the apoenzyme shift on binding one Zn11 per molecule, while resonances due to the fourth histidyl residue shift on binding of a second Zn11.583 There is one cysteinyl residue, which has also been suggested to act as a ligand, on the basis of spectroscopic studies of the Co enzyme.584... 

There are several recent reviews of the molybdenum and tungsten enzymes [4-6,23,26-36], In this chapter, we first define the metallocofactors and offer a compilation of the enzymes and their diverse activities. We then focus on the active-site structures, highlighting the confluence of crystallographic and spectroscopic studies. This is followed by a discussion of pertainent spectroscopic, structural, reactivity, and theoretical model studies. We then turn our attention to the mechanisms of catalytic activity of the molybdenum and tungsten enzymes. 

A detailed model for the enzyme mechanism has been developed by Beinert and Kennedy and their collaborators on the basis of extensive spectroscopic studies, isotope labeling, kinetic experiments, several crystal structures, and site-directed mutagenesis experiments. As usual for an enzymological work in progress, the hardness of individual aspects of the model ranges from well established to conjectural. 

---