A Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear Type 1 Copper Center with an Open Binding Site

Parisa Hosseinzadeh†, Shiliang Tian‡, Nicholas M. Marshall‡, James Hemp†, Timothy Mullen‡, Mark J. Nilges⊥, Yi-Gui Gao∇, Howard Robinson^∥, David A. Stahl^#, Robert B. Gennis^*†‡, and Yi Lu^*†‡

^†Department of Biochemistry, ^‡Department of Chemistry, ^∇Biocrystallization Facility, and ^⊥EPR Center, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States

^∥ Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000, United States

^# Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington 98195, United States

J. Am. Chem. Soc., Article ASAP

DOI: 10.1021/jacs.5b13128

Publication Date (Web): April 27, 2016

Copyright © 2016 American Chemical Society

http://pubs.acs.org/doi/abs/10.1021/jacs.5b13128 

http://pubs.acs.org/doi/pdf/10.1021/jacs.5b13128 

Abstract

Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M^–1 cm^–1) and 558 nm (2290 M^–1 cm^–1) in the UV–vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A_∥ = 98 × 10^–4 cm^–1, and another more rhombic, with a smaller A_∥ value of 69 × 10^–4 cm^–1. The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H₂O at 2.25 Å. Both UV–vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.