Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga1

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Abstract
We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH2‐terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non‐photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH‐terminal extension, which shows high homology exclusively to the COOH‐termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron‐containing holorubredoxin and zinc‐substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd‐type iron site. One‐dimensional nuclear magnetic resonance spectroscopy of recombinant Zn‐rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.