1. science
2. chemistry
3. organic chemistry

Ultrafast 2D-IR spectroscopy of nitrosylated haem-proteins using ULTRA
Contact neil.hunt@strath.ac.uk
K. Adamczyk, N. Simpson, N.T. Hunt
Department of Physics, SUPA
University of Strathclyde
C. Bellota-Anton, P.A. Hoskisson, N.P. Tucker
Strathclyde Institute of Pharmacy and Biomedical Sciences
University of Strathclyde
G.M. Greetham, M. Towrie, A.W. Parker
Central Laser Facility
STFC Rutherford Appleton Laboratory
A. Gumiero, M.A. Walsh
Diamond Light Source
Introduction
Understanding the function of biological molecules at the level
of movements of atoms or the making/breaking of chemical
bonds offers considerable potential for downstream benefits.
These range from advanced drug design strategies to the
production of synthetic, biology-inspired molecules for
technological or medical applications. The concept of the
structure-function relationship is well-established in biology but
this does not offer a complete picture of the intimate ‘chemical’
processes occurring in the active sites of biological molecules
because it neglects the role of solvent-induced and thermal
fluctuations of the protein architecture as well as the effect of
local vibrational modes. Indeed, the influence of fast protein
structural dynamics on biological processes that take place
many orders of magnitude more slowly is one of the key
questions yet to be conclusively addressed.1
structural elements in the individual steps remain the topic of
debate14,15 and enquiry. In particular, the distal side of the haem
pocket includes a histidine residue located in close proximity to
the haem centre.16-18 This residue is widely implicated in the
catalase mechanism and mutation studies have shown that its
presence is crucial to CpdI formation.19-21
The subtle way that biomolecule structure influences function is
evidenced very clearly by the haemoprotein family. This group
of proteins are responsible for a large number of biological roles
ranging from reversible ligand binding to enzymatic activity
but, to a first approximation, some of the major structural
features located near the haem centre appear to be very similar,
raising the question of exactly how the molecular architecture
influences function. An example of this can be seen in studies
showing that mutations at just four positions or fewer can
engender nitric oxide reductase or peroxidase activity upon the
ligand binding protein myoglobin. This flexibility of function
within a relatively restricted structure has led to the
haemoproteins becoming attractive templates for synthetic
systems but for this to be successful, we must first fully
understand the detailed roles of each of the main structural
elements.2-5
Ultrafast 2D-IR spectroscopy has shown great potential for
measuring the structural dynamics of biological molecules both
at the global, whole molecule level and in terms of a single
bond within the macromolecular structure by employing
vibrational probes.6-8 The purpose of this report is to summarise
recent advances of 2D-IR spectroscopy of haem proteins using
the ULTRA laser system and to demonstrate how this
technology can influence our view of the structure-function
relationship. This will be done by reference to studies of two
haem proteins: the ligand transport protein myoglobin 9 and the
catalase enzyme.10
The catalases, common to almost all aerobically-respiring
organisms, are responsible for the disproportionation of
hydrogen peroxide in a reaction that is often represented as: 11-13
catalase-Fe(III) + H2O2 → O=Fe(IV)Por+. + H2O
(1)
O=Fe(IV)Por+. + H2O2 → catalase-Fe(III) + H2O + O2
(2)
2H2O2
→
+.
2H2O + O2
(1)+(2)
where O=Fe(IV)Por is referred to as Compound 1 (CpdI). This
mechanism is widely accepted but the precise roles of catalase
Interestingly, a similarly-located and conserved distal histidine
residue is found in myoglobin. The fact that this residue is
apparently central to the functioning of two different proteins
begs questions about its role. For example, it seems reasonable
that it could be responsible for ligand binding in both cases.
This then suggests that it is the rest of the haem pocket that
controls specific functionality. Other residues in the active sites
of these proteins do differ and so must contribute to the
behaviour of the biomolecule. Most notably, the proximal
residues that coordinate with the Fe atom of the haem moiety
are different and this change could play a role in the chemical
lability of the haem ligand.22 However, it is also instructive to
ask whether the presence of the distal histidine in both
myoglobin and catalase means that the haem ligand is subject to
a similar chemical environment in both cases and it is this
question that we address here.
In each of the articles featured, the ferric form of the protein
was considered with nitric oxide bound to the haem centre
acting as a probe of the local dynamic environment.9,10 The
choice of NO arose because it binds effectively to the haem site
of Mb while the catalase enzyme is inhibited by NO binding,
meaning that it provided a stable and effective probe in both
cases. In addition, NO itself plays a fundamental role in
biology, participating in processes such as signalling and
immune responses,23-25 while higher concentrations can lead to
the deleterious effects associated with nitrosative stress. The
NO radical is also highly reactive with transition metals and
metalloproteins, such as those containing haem groups and
well-known examples include components of the respiratory
chain such as cytochrome C oxidase and key enzymes of the
tricarboxylic acid cycle such as fumarase and aconitase.26,27
Experimental
For all 2D-IR experiments, catalase and myoglobin were
contained in a pD7 deuterated phosphate buffer solution with
care taken to ensure complete H/D exchange in all cases.
MAHMA NONOate was used to nitrosylate the ferric
proteins.10 For all 2D-IR experiments, the samples were held
between two CaF2 windows separated by a 100 µm thickness
spacer.
The method for obtaining IR pump-probe and 2D-IR spectra
has been described previously; briefly, 2D-IR spectra were
acquired using the FT-2D-IR method described in Ref 10 using a
sequence of three mid-infrared (IR) laser pulses arranged in a
pseudo pump-probe beam geometry.28,29 The pulses were
generated by the ULTRA Ti:sapphire laser system pumping a
white-light seeded optical parametric amplifier (OPA) equipped
Fig 1: 2D-IR spectra of the NO stretching vibrational moode of
nitrosylated caatalase (a) and myoglobin H64Q (c). Figs (bb) and
(d) are fits of tthe data in (a) and
a (c) respectively to 2D Gauussian
lineshape funcctions.9,10
m
of the siignal and idler.. Mid
with differencce frequency mixing
IR pulses wiith a temporall duration of ~100 fs; a ceentral
frequency of 1900 cm-1 withh a bandwidth of >300 cm-1 were
employed.
Results and D
Discussion
Representativee 2D-IR spectrra for catalase and the myogglobin
(Mb) H64Q m
mutant are show
wn in Fig 1(a-d
d). In the case oof the
catalase proteiin, a single negative (red) peeak on the specctrum
diagonal wass observed annd assigned to
t the bleachh and
stimulated em
mission from the v=0-1 transition of thee NO
stretching modde of the nitrosylated protein
n (Fig 1 (a)). 100 This
was accompannied by a positivve (blue) peak shifted
s
by arounnd 30
cm-1 to lowerr probe frequeency, which was
w assigned too the
accompanyingg v=1-2 excitedd state absorptio
on.10
The 2D-IR speectrum of wild type (wt) Mb reported
r
was siimilar
to that of cattalase in that it too featured
d a single diaagonal
infrared transittion in the NO stretching regio
on. 9 By contrasst, the
2D-IR spectruum of Mb-H644Q shows two diagonal peakss (Fig
1(c)), one of w
which was locatted at the same frequency as thhat of
the wild-type protein peak and
a one that was
w shifted to llower
The structure of Mb is well-known and it is
frequency.9 T
accepted thaat wt-Mb feaatures a direect H-bondingg-type
interaction bettween the distaal histidine residue side chainn and
the haem ligannd. 30 The H64Q
Q mutation feattures replacemeent of
the distal histiidine residue with
w glutamine. The latter featuures a
more flexible side chain thaan histidine, allowing the term
minal
c
to move away
a
from the haem
functional grooup of the side chain
ligand in a fraaction of the molecules
m
in the sample. As a rresult
of this, the hiigher frequencyy peak observeed in the Mb-H
H64Q
spectrum was attributed to the
t NO stretching vibration oof the
sub-ensemble in which there was an interacttion between thhe NO
t
protein. The
T lower frequuency
ligand, similarr to the wild type
mode correspoonded to the subb-ensemble of proteins
p
withouut this
interaction.31
R spectra show
wn in Fig 1 thaat the
It is noticeablle in the 2D-IR
lineshapes of the v=0-1 and v=1-2 transiitions are elonngated
towards the diiagonal of the spectrum. The spectra for cattalase
and Mb-H64Q
Q shown were obtained
o
with a waiting time oof ~ 1
ps and this eloongation is duee to inhomogen
neous broadeniing of
the NO stretchhing vibrationall mode of both proteins. This eeffect
has been widdely reported for
fo haem proteiins and arises from
fluctuations off the electrostattic environmentt of the ligand ddue to
motion of thhe protein arcchitecture.9,10,32-34 As the prrotein
fluctuates, the effect is to varry the NO stretcching frequencyy by a
smalll amount leading to broadeninng of the transiition across thee
ensemble. In a 2D-IIR experiment at waiting timees that are shortt
in relation
r
to thee protein dynaamics that are causing thee
broaadening, this results in a diagoonal elongation
n of the 2D-IR
R
peak
ks because the sample
s
maintaiins a ‘memory’’ of the state inn
whicch it was exciteed; leading to a correlated 2D peakshape. Ass
the waiting
w
time is allowed
a
to incre
rease and becom
mes comparablee
to th
he timescales of the underl
rlying dynamiccs, the samplee
flucttuations lead to a loss of this m
memory and thee peak becomess
moree circular. Thiis so-called sppectral diffusio
on results in a
chan
nge in the profile of the 2D-IR
R peak with waaiting time andd
quan
ntification of th
he lineshape eevolution using
g fitting to 2D
D
Gausssian lineshapes (eg Fig 1 (b&d))9,10 gives rise to ann
expo
onential-type decay
d
with thhe timescales reporting thee
dynaamics of the frequency-freqquency correlation functionn
(FFC
CF) of the NO vibration,
v
which
ch in turn reportt on the proteinn
dynaamics influencin
ng the ligand.9,335-39
The FFCFs extracted from the 2D
D-IR data for catalase,
c
wt-Mbb
and Mb-H64Q arre shown in FFig 2.9,10 In each case ann
expo
onential function was shown too represent the data
d well (solidd
liness) and it is interesting to note th
the similarities and differencess
in th
he data across the three proteinns. Specifically
y, both catalasee
and the wt-Mb sh
howed a fast ddynamic comp
ponent (~3 ps))
o
The stattic parameter was
w assigned too
alongside a static offset.
w motions caussing broadeninng of the NO transition butt
slow
whicch were too slow
w to be captureed by a 2D-IR experiment
e
thatt
was temporally lim
mited by the viibrational lifetiime of the NO
O
n. Although ccatalase and wt-Mb
w
showedd
stretching vibration
similar fast dynamics, the static ccomponent in Mb was large,,
v
small in ccatalase. This sh
howed that thee
wherreas this was very
motiions causing brroadening are complete within 20-30 ps inn
catallase while tho
ose in Mb perrsist for much
h longer. Thiss
obseervation was ussed to infer a ddynamically more
m
constrictedd
struccture in catalasee. 10
In asssigning the fasst dynamics, it was noted thaat while the wt-Mb and the H64Q
Q mutation both
th showed sim
milar slow/staticc
c
in th
the wt-Mb dataa was replicatedd
dynaamics, the fast component
only
y for the sub-ensemble of the H
H64Q mutation
n with a similarr
vibraational frequency to the wt prrotein. 9 This fast
f componentt
was absent in the su
ub-ensemble off Mb-H64Q thaat had no directt
d
side chaain and the NO
O ligand. Thiss
link between the distal
allow
wed assignmen
nt of the faast dynamics of wt-Mb too
interraction between
n the distal histid
idine and the haaem ligand. 9
Giveen the similaritiies in both distaal pocket archittecture and fastt
dynaamics between
n wt-Mb andd catalase, an
a anaologouss
assig
gnment of the fast dynamics of catalase to an interactionn
betw
ween the distal histidine aand the NO would seem
m
apprropriate. Howev
ver, it was repoorted that the crystal
c
structuree
of the
t
nitrosylated protein was
as more consiistent with ann
interraction of NO with
w a conservedd bound water molecule
m
in thee
distaal pocket. Whille this would seeem contradicttory, the boundd
wateer molecule wass also hydrogenn-bonded to the distal histidinee
in su
uch a way as to
t communicatee the fast dynaamics from thee
proteein scaffold to the haem lligand. Thus, the dynamicss
obseerved in catalasee were comparaable to those off wt-Mb but thee
mech
hanism by which they were oobserved was different
d
for thee
two proteins. It was further hypotthesised that th
his bound waterr
moleecule, in conjun
nction with a nnetwork of otheers observed inn
the crystal
c
structure, were the oriigins of the strructurally moree
conffined active sitee and so the smaall size of the static
s
parameterr
in th
he catalase FFCF
F. 10
In su
ummary, by comparing these sets of results,, it can be seenn
that 2D-IR providess insight into thhe local chemiccal environmentt
of th
he haem ligand
d in both a liggand transport protein and ann
enzy
yme. Furtherm
more, cross-diisciplinary intteraction withh
strucctural biologistts enables this data to be in
nterpreted in a
physsically-meaning
gful manner forr biological applications. Thee
concclusions suggest that the ligannd transport pro
otein features a
moree flexible struccture with an iinteraction betw
ween the distall
pock
ket and ligand that presumaably serves to aid reversiblee
binding of a diiatomic ligand. In contrast, thee enzyme locatees the
ligand in a moore constrained geometry conssistent with thee need
to access a pparticular transiition state as part
p
of the reaaction
mechanism. T
Thus, although the
t structures may
m seem similaar, the
natures of the active sites of these
t
two proteiins differ markeedly.
Acknowledgeements
Support for thhis work is acknnowledged from
m the STFC Ceentral
Laser Facilityy, the Europeean Research Council (2022706),
EPSRC (EP/J000975X/1) and the Leverhulme Trust (RPG-2248).
Fig 2: FFCF data for nitrossylated catalasee (a) and myogglobin
m Ref9
(b) extracted ffrom 2D-IR datta.9,10 Figure reeproduced from
- Reproduced by permission of
o the PCCP Ow
wner Societies
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