Insertion of tetrapyrrole cofactors into cytochrome cd1 nitrite reductase
Final Report Abstract
In this project, the maturation of the periplasmic cytochrome cd1 nitrite reductase NirS was studied. The initial hypothesis was that the periplasmic protein NirF catalyzes the last step of heme d1 biosynthesis and that the c-type cytochrome NirN acts as a heme d1 chaperone, which inserts the cofactor into NirS. During the first PROTRAIN funding period, it was shown that NirF from Pseudomonas aeruginosa carries a lipid anchor that attaches the protein to the periplasmic side of the inner membrane. Moreover, it was found that all three proteins, NirF, NirN and NirS, interact with each other in vivo. However, at the beginning of the second funding period, the precise roles of NirF and NirN during NirS maturation were still unclear and had to be elucidated. Within the second funding period, we finally established the role of NirN. This protein acts as the terminal enzyme of heme d1 biosynthesis converting dihydroheme d1 to heme d1. Thus, NirN plays the role that previously had been reserved for NirF. This result was very surprising, and at the same time represented a huge progress toward a better understanding of NirS maturation. The reaction catalyzed by NirN was further characterized. We found that NirN represents a rather unusual dehydrogenase, which uses the one-electron acceptor heme c in order to catalyze a two-electron oxidation reaction. This is possible, because one of the electrons is transferred to the heme c and the second electron remains on the central iron ion of the reaction product heme d1. Further, we demonstrated that NirN transfers the newly formed heme d1 to semi-apo NirS. The crystallization of NirN yielded first crystals, which have to be optimized in the future in order to solve the 3D structure of the protein. For NirF we envisaged a new role during NirS maturation, namely that of a periplasmic dihydroheme d1 uptake protein that is involved in the transport of the heme d1 precursor across the cytoplasmic membrane. According to this idea, the ability of NirF to bind dihydroheme d1 was tested. We found that NirF is indeed able to bind dihydroheme d1 without converting it to heme d1. The crystallization of NirF yielded first crystals, which will be optimized in the future for structure determination.
Publications
-
(2013) Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF. Biosci. Rep., 33, art:e00048
Nicke, T., Schnitzer, T., Münch, K., Adamczack, J., Haufschildt, K., Buchmeier, S., Kucklick, M., Felgenträger, U., Jänsch, L., Riedel, K. and Layer, G.
-
(2014) NirN from Pseudomonas aeruginosa is a novel electron-bifurcating dehydrogenase catalyzing the last step of heme d1 biosynthesis. J. Biol. Chem., 289, 30753-30762
Adamczack, J., Hoffmann, M., Papke, U., Haufschildt, K., Nicke, T., Bröring, M., Sezer, M., Weimar, R., Kuhlmann, U., Hildebrandt, P. and Layer, G.
