Regulating the nitrite reductase activity of myoglobin by redesigning the heme active center

Abstract

Heme proteins perform diverse functions in living systems, of which nitrite reductase (NIR) activity receives much attention recently. In this study, to better understand the structural elements responsible for the NIR activity, we used myoglobin (Mb) as a model heme protein and redesigned the heme active center, by introducing one or two distal histidines, and by creating a channel to the heme center with removal of the native distal His64 gate (His to Ala mutation). UV-Vis kinetic studies, combined with EPR studies, showed that a single distal histidine with a suitable position to the heme iron, i.e., His43, is crucial for nitrite (NO2(-)) to nitric oxide (NO) reduction. Moreover, creation of a water channel to the heme center significantly enhanced the NIR activity compared to the corresponding mutant without the channel. In addition, X-ray crystallographic studies of F43H/H64A Mb and its complexes with NO2(-) or NO revealed a unique hydrogen-bonding network in the heme active center, as well as unique substrate and product binding models, providing valuable structural information for the enhanced NIR activity. These findings enriched our understanding of the structure and NIR activity relationship of heme proteins. The approach of creating a channel in this study is also useful for rational design of other functional heme proteins.

Rational design of metalloenzymes: From single to multiple active sites

“…14a), with the activity of mesoMb ~3.4-fold that of WT Mb. On the other hand, Lin and coworkers [135] found that the NIR activity of Mb can be regulated by redesign of the heme active center instead of modifying the heme group. By studying a series of Mb distal histidine mutants and the channel mutants, it showed that an enhancement of NIR activity was achieved by creation of a water channel to the heme center (H64A mutation) and relocation of the distal histidine (F43H mutation) (Fig.…”

“…Copyright 2016 Elsevier; (b) Relative reactivities of Mb distal histidine mutants and the channel mutants with respect to that of WT Mb. The X-ray structure of met F43H/H64A Mb was shown for clarification[135].…”

Nitrite Reduction in Aqueous Solution Mediated by Amavadin Homologues: N2O Formation and Water Oxidation

“…Various fungi, including A. muscaria,a lso contribute to the nitrogen cycle with nitrite as possible metabolic substrate. Additionally,r ational design based on metallobiomolecules [62][63][64] hasb een employed for as imilar purpose. [21] Despite such great importance,t he chemical homogeneous reduction of nitrites by inorganic transition metals and internal transition-metal compounds has rarely been investigated.M ost studies focusedo ni ron [33][34][35][36][37][38][39][40][41][42][43][44][45] or copper [46][47][48][49][50][51][52] complexes that mimicked nitrite reductases.H owever,N O 2 À reduction with Ru, [53] Mo, [54][55][56] Ti,C r, [56] Co [57][58][59] andU [60] was also reported.…”

Design of artificial metalloproteins/metalloenzymes by tuning noncovalent interactions

“…In a related study, Lin, Tan, and co-workers created a channel to the heme pocket of Mb by removing distal His64, a gate for O2 binding, and introducing a distal histidine at position 43 (F43H/H64A) [103]. A unique water channel was formed between the heme pocket and the solvent by successive H-bonding interactions of water molecules (W1-W7) in F43H/H64A Mb (Fig.…”