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@ARTICLE{Martini:471645,
author = {Martini, Maria and Ruediger, Olaf and Breuer, Nina and
Nöring, Birgit and DeBeer, Serena and Rodriguez Macia,
Patricia and Birrell, James},
editor = {Bren, Kara},
title = {{T}he {N}onphysiological {R}eductant {S}odium {D}ithionite
and [{F}e{F}e] {H}ydrogenase: {I}nfluence on the {E}nzyme
{M}echanism},
journal = {Journal of the American Chemical Society},
volume = {143},
number = {43},
issn = {0002-7863},
address = {Washington, DC},
publisher = {American Chemical Society},
reportid = {PUBDB-2021-04513, I-20200084},
pages = {18159 - 18171},
year = {2021},
abstract = {[FeFe] hydrogenases are highly active enzymes for
interconverting protons and electrons with hydrogen (H$_2$).
Their active site H-cluster is formed of a canonical
[4Fe-4S] cluster ([4Fe-4S]$_H$) covalently attached to a
unique [2Fe] subcluster ([2Fe]$_H$), where both sites are
redox active. Heterolytic splitting and formation of H$_2$
takes place at [2Fe]$_H$, while [4Fe-4S]$_H$ stores
electrons. The detailed catalytic mechanism of these enzymes
is under intense investigation, with two dominant models
existing in the literature. In one model, an alternative
form of the active oxidized state H$_{ox}$, named H$_{ox}$H,
which forms at low pH in the presence of the
nonphysiological reductant sodium dithionite (NaDT), is
believed to play a crucial role. H$_{ox}$H was previously
suggested to have a protonated [4Fe-4S]$_H$. Here, we show
that H$_{ox}$H forms by simple addition of sodium sulfite
(Na$_2$SO$_3$, the dominant oxidation product of NaDT) at
low pH. The low pH requirement indicates that sulfur dioxide
(SO$_2$) is the species involved. Spectroscopy supports
binding at or near [4Fe-4S]$_H$, causing its redox potential
to increase by ∼60 mV. This potential shift detunes the
redox potentials of the subclusters of the H-cluster,
lowering activity, as shown in protein film electrochemistry
(PFE). Together, these results indicate that H$_{ox}$H and
its one-electron reduced counterpart H$_{red}$'H are
artifacts of using a nonphysiological reductant, and not
crucial catalytic intermediates. We propose renaming these
states as the "dithionite (DT) inhibited" states
H$_{ox}$-DT$_i$ and H$_{red}$-DT$_i$. The broader potential
implications of using a nonphysiological reductant in
spectroscopic and mechanistic studies of enzymes are
highlighted.},
cin = {DOOR ; HAS-User / MPG},
ddc = {540},
cid = {I:(DE-H253)HAS-User-20120731 / I:(DE-H253)MPG-20120806},
pnm = {6G3 - PETRA III (DESY) (POF4-6G3) / FS-Proposal: I-20200084
(I-20200084)},
pid = {G:(DE-HGF)POF4-6G3 / G:(DE-H253)I-20200084},
experiment = {EXP:(DE-H253)P-P01-20150101},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:34668697},
UT = {WOS:000715845900034},
doi = {10.1021/jacs.1c07322},
url = {https://bib-pubdb1.desy.de/record/471645},
}