Quantification of nitrogenase in Trichodesmium IMS 101: Implications for iron limitation of nitrogen fixation intheocean

Iron is widely thought to limit nitrogen fixation in the open, oligotrophic ocean due to the low solubility of Fe in oxic seawater and the high Fe demand for the nitrogenase holozyme. However, empirical evidence for Fe limitation of field populations of Trichodesmium based on either incubation experiments or molecular and physiological indicators has not quantitatively related Fe supply to the cellular Fe quotas for nitrogenase. Rather, the Fe required for N₂ fixation has been inferred from in vivo catalytic activity. Using a pet14b expression vector, we cloned the nif H gene (encoding the Fe-protein, which contains 4Fe atoms per subunit) from Trichodesmium IMS 101, and purified the Histagged apoprotein with which we derived a primary standard based on quantitative Western blots. Using a standard curve derived from the cloned Trichodesmium Fe apoprotein, we measured the absolute abundance of the Fe-protein in iron-replete cultures of this marine diazotroph. At peak expression, we calculate 0.04 mg nitrogenase mg^-1 C.Assuming a conservative stoichiometry of two Fe-protein subunits per MoFe protein (which contains 15 Fe atoms per subunit, or a total of 38 atoms of Fe per holozyme), we estimate 236 μmolFeisboundtonitrogenaseper mol cellularC. This estimate is about 10 times greater than the Fe previously calculated to support diazotrophic growth under these conditions. Our results suggest that under bloom conditions in the subtropical North Atlantic and North Pacific, as much as ~2.22 and 0.06 μmol m^-3 of Fe is bound to Trichodesmium nitrogenase respectively. Such a high quota represents between ~50% and > 100% summer-time average particulate Fe in surface waters, suggesting the importance of this taxon for the retention and biogeochemical cycling of Fe. Moderate growth (0.10 day^-1) towards the end of these blooms would require a vertical flux as high as ~23 μmol Fe day^-1 m^-2 into the mixed layer.