Expression, Purification, and Activity Analysis of Chlorophyllide Oxidoreductase and Ni2+-Sirohydrochlorin a,c-Diamide Reductase

Abstract

Nitrogenase-like enzymes play a vital role in the reduction of the conjugated ring systems of diverse tetrapyrrole molecules. The biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR); whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni²⁺-sirohydrochlorin a,c-diamide in the course of coenzyme F₄₃₀ biosynthesis. The first part of this chapter describes the production and purification of the COR components (BchY/BchZ)₂ and BchX₂, the measurement of COR activity, and the trapping of the ternary COR complex; and the second part describes the strategy for obtaining homogenous and catalytically active preparations of CfbC₂ and CfbD₂ and a suitable method for extracting the reaction product Ni²⁺-hexahydrosirohydrochlorin a,c-diamide.

Keywords: Chlorophyll biosynthesis; Chlorophyllide oxidoreductase (COR); Coenzyme F430 biosynthesis; Dynamic switch protein; Ni2+-sirohydrochlorin a,c-diamide reductase (CfbC/D); Nitrogenase-like enzymes.