Expression, purification, crystallization and preliminary X ray structure analysis of wild type and L M196 H mutant Rhodobacter sphaeroides reaction centres

The electron and proton transport mediated by protein bound cofactors in photosynthesis have been investigated by various methods in order to determine the energetics, the dynamics and the pathway of this process. In purple bacteria, primary photosynthetic charge separation and the build up of a proton gradient across the periplasmic membrane are catalyzed by the photosynthetic reaction centre RC . Here, the purification, crystallization and preliminary X ray analysis of wild type and L M196 H mutant RCs of Rhodobacter sphaeroides are presented, enabling study of the influence of the protein environment of the primary electron donor on the spectral properties and photochemical activity of the R

Incorporation of Copper Ions into T2 T3 Centers of Two Domain Laccases

Laccase belongs to the family of copper containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2 T3 centers of recombinant two domain laccase Streptomyces griseoflavus Ac 993. The occupancy of the T3 center by copper ions was found to increase with an increasing copper content in the culture medium and after dialysis of the protein preparation against a copper sulfate containing buffer. The T2 center was filled only when overproducer strain cells were grown at a higher copper concentration in the medium. Two domain laccases were assumed to possess a channel that serves to deliver copper ions to the T3 center during the formation of the three dimensional laccase conformation and dialysis of the protein preparation. A narrower channel leads to the T2 center in two domain laccases compared with three domain ones, rendering the center less accessible for copper atoms. The incorporation of copper ions into the T2 center of two domain laccases is likely to occur in the course of their biosynthesis or the formation of a functional trime