Substrate binding spectroscopic changes upon

The P450 reaction cycle (Scheme 10.4) starts with four stable intermediates that have been characterized by spectroscopic methods. The resting state of the enzyme is a six-coordinate, low-spin ferric state (complex I) with water (or hydroxide) coordinated trans to the cysteinate ligand. The spin state of the iron changes to high-spin upon substrate binding and results in a five-coordinate ferric ion (com-... 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

In a spectroelectrochemical experiment at modified gold for substrate-bound P450cam an of — 373 mV was found. The surface interaction and direct electrochemical transformation does not affect the enzyme structure as was confirmed spectroscopically. Both, upon direct electrochemical reduction and upon ligand binding the spectral changes clearly indicated the native state of P450cam during reversible reduction and oxidation [181]. 

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