Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome <i>c</i> Nitrite Reductase (NrfA)
Victor Sosa Alfaro, Julius Campeciño, Matthew Tracy, Sean J. Elliott, Eric L. Hegg, Nicolai Lehnert
Abstract
NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.