CHAPTER FOUR Escherichia coli Cytochrome c Nitrite Reductase NrfA Thomas A. Clarke,* Paul C. Mills,* Susie R. Poock,* Julea N. Butt,* ,† Myles R. Cheesman, † Jeffrey A. Cole, ‡ Jay C. D. Hinton, § Andrew M. Hemmings,* ,† Gemma Kemp, † Christopher A. G. So ¨derberg,* Stephen Spiro, k Jessica Van Wonderen, † and David J. Richardson* Contents 1. Introduction 64 2. Measurement of Cytochrome c Nitrite Reductase-Dependent Consumption of Nitric Oxide in Whole Cells 66 3. Growth of E. coli Optimized for Cytochrome c Nitrite Reductase Production for Use in Enzyme Purification 66 4. Purification of Cytochrome c Nitrite Reductase 68 5. Assaying the Cytochrome c Nitrite Reductase 69 6. Crystallization of E. coli Cytochrome c Nitrite Reductase 73 7. Concluding Remarks 74 Acknowledgments 76 References 76 Abstract The periplasmic cytochrome c nitrite reductase (Nrf ) system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. Nitric oxide (NO) is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate. This chapter describes the method used to assay Nrf-catalyzed NO reduction in whole cells of E. coli and the procedures for preparing highly purified NrfA suitable for use in kinetic, spectroscopic, voltammetric, and crystallization studies. Methods in Enzymology, Volume 437 # 2008 Elsevier Inc. ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)37004-3 All rights reserved. * Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich, United Kingdom { School of Biological Sciences, University of East Anglia, Norwich, United Kingdom { School of Biosciences, University of Birmingham, Edgbaston, Birmingham } Institute of Food Research, Norwich, United Kingdom k Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 63