Rapid scanning spectroscopy intermediates

Experimental limitations initially limited the types of molecular systems that could be studied by TRIR spectroscopy. The main obstacles were the lack of readily tunable intense IR sources and sensitive fast IR detectors. Early TRIR work focused on gas phase studies because long pathlengths and/or multipass cells could be used without interference from solvent IR bands. Pimentel and co-workers first developed a rapid scan dispersive IR spectrometer (using a carbon arc broadband IR source) with time and spectral resolution on the order of 10 ps and 1 cm , respectively, and reported the gas phase IR spectra of a number of fundamental organic intermediates (e.g., CH3, CD3, and Cp2). Subsequent gas phase approaches with improved time and spectral resolution took advantage of pulsed IR sources. 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

Silverman s studies on mechanism based MAO inactivation have provided overwhelming support for the role of electron transfer in the MAO catalyzed dealkylation of amines. It must be mentioned however that spectroscopic attempts for detecting the radical ion intermediates have hitherto been unsuccessful. Yasanobu and coworkers could not find EPR spectral evidence for radical intermediates in MAO-catalyzed oxidation of benzylamine [205]. Miller et al. failed to observe the flavin semiquinone or an amine-flavin adduct in rapid-scan-stopped flow spectroscopy [206]. The only time-dependent absorption change observed in this study was the bleaching of the oxidized flavin. Furthermore, no influence of a magnetic field up to 6500 G was observed on the rate of MAO B reduction. The reaction rates of systems with kinetically significant radical pair intermediates are known to be altered... 

Diffusion of NO into the distal pocket is considered to be the rate-limiting step. An intermediate, [HbnlOONO], in reaction 7.32 has been proposed on the basis of rapid-scan UV-Vis spectroscopy and EPR evidence.92 The mechanism of decay of the peroxynitrite intermediates is still a matter of debate.89-91,93 An alternative route to isomerization implies the homolysis of the 0-0 bond in [HbnlOONO], leading to N02 and FeIV0-protein intermediates (Figure 7.20). 

If the investigation of the chemical mechanisms of enzyme-catalyzed reactions is to be successful, then detection and identification of transient intermediates along the reaction path are essential components. The application of rapid-scanning stopped-flow (RSSF) UV/visible spectroscopy to the study of enzyme catalysis may be of considerable significance in certain instances as a tool for both the direct detection and the identification of transient chemical intermediates. 

---