Colin W. J. Lockwood †, Bénédicte Burlat †, Myles R. Cheesman †, Melanie Kern §, Jörg Simon §, Thomas A. Clarke ‡, David J. Richardson ‡, and Julea N. Butt *†‡
†Centre
for Molecular and Structural Biochemistry, School of Chemistry
and ‡School
of Biological Sciences, University of East
Anglia, Norwich Research
Park, Norwich, NR4 7TJ, U.K.
§ Microbial
Energy Conversion and Biotechnology, Department of Biology, Technische Universität Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt, Germany
J. Am. Chem. Soc., Article ASAP
DOI: 10.1021/ja512941j
Publication Date (Web): February 6, 2015
Copyright © 2015 American Chemical Society
Abstract
Cytochrome c
nitrite reductases perform a key step in the biogeochemical N-cycle by
catalyzing the six-electron reduction of nitrite to ammonium. These
multiheme cytochromes contain a number of His/His ligated c-hemes
for electron transfer and a structurally differentiated heme that
provides the catalytic center. The catalytic heme has proximal ligation
from lysine, or histidine, and an exchangeable distal ligand bound
within a pocket that includes a conserved histidine. Here we describe
properties of a penta-heme cytochrome c nitrite reductase in
which the distal His has been substituted by Asn. The variant is unable
to catalyze nitrite reduction despite retaining the ability to reduce a
proposed intermediate in that process, namely, hydroxylamine. A
combination of electrochemical, structural and spectroscopic studies
reveals that the variant enzyme simultaneously binds nitrite and
electrons at the catalytic heme. As a consequence the distal His is
proposed to play a key role in orienting the nitrite for N–O bond
cleavage. The electrochemical experiments also reveal that the distal
His facilitates rapid nitrite binding to the catalytic heme of the
native enzyme. Finally it is noted that the thermodynamic descriptions
of nitrite- and electron-binding to the active site of the variant
enzyme are modulated by the prevailing oxidation states of the His/His
ligated hemes. This behavior is likely to be displayed by other
multicentered redox enzymes such that there are wide implications for
considering the determinants of catalytic activity in this important and
varied group of oxidoreductases.
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