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Abstract: The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c 3 was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M21 and M181. The rubredoxin binding surface in the complex with cytochrome c 3 was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c 3 are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in 1HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes. [Copyright &y& Elsevier]

Marinobacter hydrocarbonoclasticus nitric oxide reductase, c NOR, is an integral membrane protein composed of two subunits with different roles, NorC (electron transfer) and NorB (catalytic) that receives electrons from the soluble cytochrome c 552 and reduces nitric oxide to nitrous oxide in the denitrification pathway. The solvent-exposed domain of NorC, harboring a c -type heme was heterologously produced, along with its physiological electron donor, cytochrome c 552 . These two proteins were spectroscopically characterized and shown to be similar to the native proteins, both being low-spin and Met-His coordinated, with the soluble domain of NorC presenting some additional features of a high-spin heme, which is consistent with the higher solvent accessibility of its heme and weaker coordination of the methionine axial ligand. The electron transfer complex between the two proteins has a 1:1 stoichiometry, and an upper limit for the dissociation constant was estimated by 1 H NMR titration to be 1.2 ± 0.4 μM. Electrochemical techniques were used to characterize the interaction between the proteins, and a model structure of the complex was obtained by molecular docking. The electrochemical observations point to the modulation of the NorC reduction potential by the presence of NorB, tuning its ability to receive electrons from cytochrome c 552 . [ABSTRACT FROM AUTHOR]