Biochemical and spectroscopic characterization of dinuclear Mn-sites in artificial four-helix bundle proteins

• Published in: Biochimica et biophysica acta. Bioenergetics 2017-12, Vol.1858 (12), p.945-954
• Main Authors: Olson, Tien L., Espiritu, Eduardo, Edwardraja, Selvakumar, Canarie, Elizabeth, Flores, Marco, Williams, JoAnn C., Ghirlanda, Giovanna, Allen, James P.
• Format: Article
• Language: English
• Subjects: Binding Sites; Catalase; Circular Dichroism; De novo design; Electron Spin Resonance Spectroscopy; Electron transfer; Hydrogen Peroxide - chemistry; Hydrogen Peroxide - metabolism; Manganese - chemistry; Metalloproteins; Metalloproteins - chemical synthesis; Metalloproteins - chemistry; Metalloproteins - metabolism; Models, Molecular; Oxygen - chemistry; Photosynthesis; Protein Binding; Protein Conformation, alpha-Helical
• ISSN: 0005-2728; 1879-2650
• DOI: 10.1016/j.bbabio.2017.08.013

To better understand metalloproteins with Mn-clusters, we have designed artificial four-helix bundles to have one, two, or three dinuclear metal centers able to bind Mn(II). Circular dichroism measurements showed that the Mn-proteins have substantial α-helix content, and analysis of electron paramagnetic resonance spectra is consistent with the designed number of bound Mn-clusters. The Mn-proteins were shown to catalyze the conversion of hydrogen peroxide into molecular oxygen. The loss of hydrogen peroxide was dependent upon the concentration of protein with bound Mn, with the proteins containing multiple Mn-clusters showing greater activity. Using an oxygen sensor, the oxygen concentration was found to increase with a rate up to 0.4μM/min, which was dependent upon the concentrations of hydrogen peroxide and the Mn-protein. In addition, the Mn-proteins were shown to serve as electron donors to bacterial reaction centers using optical spectroscopy. Similar binding of the Mn-proteins to reaction centers was observed with an average dissociation constant of 2.3μM. The Mn-proteins with three metal centers were more effective at this electron transfer reaction than the Mn-proteins with one or two metal centers. Thus, multiple Mn-clusters can be incorporated into four-helix bundles with the capability of performing catalysis and electron transfer to a natural protein. [Display omitted] •A four-helix framework supports multiple dinuclear manganese clusters.•Dinuclear Mn-clusters catalyze conversion of hydrogen peroxide to molecular oxygen.•Artificial Mn-proteins transfer electrons to light-oxidized bacteriochlorophylls.