Analysis of metal incorporation during overexpression of Clostridium pasteurianum rubredoxin by electrospray FTICR mass spectrometry

The rate of production of Clostridium pasteurianum rubredoxin overexpressed in Escherichia coli was examined by electrospray ionization-Fourier transf orm ion cyclotron resonance (ESI-FTICR) mass spectrometry. Previous work ha d shown that this heterologous expression resulted in isolation of both iro n-containing (FeRd) and zinc-containing (ZnRd) rubredoxins. In the present work, minimally processed cell lysates of E. coli were analyzed in order to monitor the production of FeRd and ZnRd. The sensitivity of the measuremen t favored FeRd relative to ZnRd, and this differential sensitivity was quan titated using previously separated and purified rubredoxins. A time course study indicated that ZnRd and FeRd are produced simultaneously during overe xpression, but at different rates. The ratio of the concentration of ZnRd t o FeRd increased in a linear fashion during 3 h following induction of over expression. Since only FeRds have been reported from native bacteria and ar chaea, the data suggest that either Zn2+ is sequestered from rubredoxins du ring native biosynthesis or that ZnRds may have escaped detection in the na tive microorganisms. ESI-FTICR mass spectrometry is shown to be a useful to ol for monitoring metal insertion during protein biosynthesis.