Expression, Purification, and Activity Analysis of Chlorophyllide Oxidoreductase and Ni 2+-Sirohydrochlorin a,c-Diamide Reductase

Methods Mol Biol. 2019;1876:125-140. doi: 10.1007/978-1-4939-8864-8_8.


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.

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / isolation & purification
  • Alcohol Oxidoreductases / metabolism
  • Catalytic Domain
  • Chlorophyll / biosynthesis
  • Metalloporphyrins / metabolism
  • Metalloproteins / chemistry
  • Metalloproteins / isolation & purification*
  • Metalloproteins / metabolism*
  • Multienzyme Complexes
  • Nickel / chemistry
  • Oxidation-Reduction
  • Uroporphyrins / chemistry*


  • Metalloporphyrins
  • Metalloproteins
  • Multienzyme Complexes
  • Uroporphyrins
  • Chlorophyll
  • sirohydrochlorin
  • factor F430
  • Nickel
  • Alcohol Oxidoreductases