15 research outputs found
Small-angle X-ray scattering studies of enzymes
Enzyme function requires conformational changes to achieve substrate binding, domain rearrangements, and interactions with partner proteins, but these movements are difficult to observe. Small-angle X-ray scattering (SAXS) is a versatile structural technique that can probe such conformational changes under solution conditions that are physiologically relevant. Although it is generally considered a low-resolution structural technique, when used to study conformational changes as a function of time, ligand binding, or protein interactions, SAXS can provide rich insight into enzyme behavior, including subtle domain movements. In this perspective, we highlight recent uses of SAXS to probe structural enzyme changes upon ligand and partner-protein binding and discuss tools for signal deconvolution of complex protein solutions
The Elusive 5ā²-Deoxyadenosyl Radical: Captured and Characterized by EPR and ENDOR Spectroscopies
The 5\u27-deoxyadenosyl radical (5\u27-dAdoā¢) abstracts a substrate H-atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5\u27-dAdoā¢ has eluded characterization despite efforts spanning more than a half-century. Here we report generation of 5\u27-dAdoā¢ in a RS enzyme active site at 12 K, using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S]+cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S-C\u27 bond cleavage
A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF
[FeFe]-hydrogenases are natureās
most prolific hydrogen
catalysts, excelling at facilely interconverting H<sub>2</sub> and
protons. The catalytic core common to all [FeFe]-hydrogenases is a
complex metallocofactor, referred to as the H-cluster, which is composed
of a standard [4Fe-4S] cluster linked through a bridging thiolate
to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide,
and cyanide ligands. This 2Fe subcluster is synthesized and inserted
into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF,
and HydG. HydE and HydG are radical <i>S</i>-adenosylmethionine
enzymes and synthesize the nonprotein ligands of the H-cluster. HydF
is a GTPase that functions as a scaffold or carrier for 2Fe subcluster
production. Herein, we utilize UVāvisible, circular dichroism,
and electron paramagnetic resonance spectroscopic studies to establish
the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound
to HydF. We have used spectroelectrochemical titrations to assign
ironāsulfur cluster midpoint potentials, have shown that HydF
purifies with a reduced [2Fe-2S] cluster in the absence of exogenous
reducing agents, and have tracked ironāsulfur cluster spectroscopic
changes with quaternary structural perturbations. Our results provide
an important foundation for understanding the maturation process by
defining the ironāsulfur cluster content of HydF prior to its
interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster
of HydF either acts as a placeholder for HydG-derived FeĀ(CO)<sub>2</sub>CN species or serves as a scaffold for 2Fe subcluster assembly
Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5ā²-dAdoā¢ āFree Radicalā Is Never Free
Lysine 2,3-aminomutase (LAM) is a
radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme
and, like other members of this superfamily,
LAM utilizes radical-generating machinery comprising SAM anchored
to the unique Fe of a [4Fe-4S] cluster via a classical five-membered
N,O chelate ring. Catalysis is initiated by reductive cleavage of
the SAM SāC5ā² bond, which creates the highly reactive
5ā²-deoxyadenosyl radical (5ā²-dAdoā¢), the same
radical generated by homolytic CoāC bond cleavage in B<sub>12</sub> radical enzymes. The SAM surrogate <i>S</i>-3ā²,4ā²-anhydroadenosyl-l-methionine (anSAM) can replace SAM as a cofactor in the isomerization
of l-Ī±-lysine to l-Ī²-lysine by LAM,
via the stable allylic anhydroadenosyl radical (anAdoā¢). Here
electron nuclear double resonance (ENDOR) spectroscopy of the anAdoā¢
radical in the presence of <sup>13</sup>C, <sup>2</sup>H, and <sup>15</sup>N-labeled lysine completes the picture of how the active
site of LAM from <i>Clostridium subterminale</i> SB4 ātamesā the 5ā²-dAdoā¢ radical,
preventing it from carrying out harmful side reactions: this āfree
radicalā in LAM is never free. The low steric demands of the
radical-generating [4Fe-4S]/SAM construct allow the substrate target
to bind adjacent to the SāC5ā² bond, thereby enabling
the 5ā²-dAdoā¢ radical created by cleavage of this bond
to react with its partners by undergoing small motions, ā¼0.6
Ć
toward the target and ā¼1.5 Ć
overall, that are
controlled by tight van der Waals contact with its partners. We suggest
that the accessibility to substrate and ready control of the reactive
C5ā² radical, with āvan der Waals controlā of
small motions throughout the catalytic cycle, is common within the
radical SAM enzyme superfamily and is a major reason why these enzymes
are the preferred means of initiating radical reactions in nature
Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation
Nature utilizes [FeFe]-hydrogenase
enzymes to catalyze the interconversion
between H<sub>2</sub> and protons and electrons. Catalysis occurs
at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated
2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis
of this unique metallocofactor is accomplished by three maturase enzymes
denoted HydE, HydF, and HydG. HydE and HydG belong to the radical <i>S</i>-adenosylmethionine superfamily of enzymes and synthesize
the nonprotein ligands of the H-cluster. These enzymes interact with
HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe
subcluster assembly. Prior characterization of HydF demonstrated the
protein exists in both dimeric and tetrameric states and coordinates
both [4Fe-4S]<sup>2+/+</sup> and [2Fe-2S]<sup>2+/+</sup> clusters
[Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick,
J. B. (2016) <i>Biochemistry 55</i>, 3514ā3527].
Herein, electron paramagnetic resonance (EPR) is utilized to characterize
the [2Fe-2S]<sup>+</sup> and [4Fe-4S]<sup>+</sup> clusters bound to
HydF. Examination of spin relaxation times using pulsed EPR in HydF
samples exhibiting both [4Fe-4S]<sup>+</sup> and [2Fe-2S]<sup>+</sup> cluster EPR signals supports a model in which the two cluster types
either are bound to widely separated sites on HydF or are not simultaneously
bound to a single HydF species. Gel filtration chromatographic analyses
of HydF spectroscopic samples strongly suggest the [2Fe-2S]<sup>+</sup> and [4Fe-4S]<sup>+</sup> clusters are coordinated to the dimeric
form of the protein. Lastly, we examined the 2Fe subcluster-loaded
form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase
activation. Together, the results indicate a specific role for the
HydF dimer in the H-cluster biosynthesis pathway
EPR and FTIR Analysis of the Mechanism of H<sub>2</sub> Activation by [FeFe]-Hydrogenase HydA1 from Chlamydomonas reinhardtii
While a general model
of H<sub>2</sub> activation has been proposed
for [FeFe]-hydrogenases, the structural and biophysical properties
of the intermediates of the H-cluster catalytic site have not yet
been discretely defined. Electron paramagnetic resonance (EPR) spectroscopy
and Fourier transform infrared (FTIR) spectroscopy were used to characterize
the H-cluster catalytic site, a [4Fe-4S]<sub>H</sub> subcluster linked
by a cysteine thiolate to an organometallic diiron subsite with CO,
CN, and dithiolate ligands, in [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii (CrHydA1). Oxidized CrHydA1
displayed a rhombic 2.1 EPR signal (<i>g</i> = 2.100, 2.039,
1.997) and an FTIR spectrum previously assigned to the oxidized H-cluster
(H<sub>ox</sub>). Reduction of the H<sub>ox</sub> sample with 100%
H<sub>2</sub> or sodium dithionite (NaDT) nearly eliminated the 2.1
signal, which coincided with appearance of a broad 2.3ā2.07
signal (<i>g</i> = 2.3ā2.07, 1.863) and/or a rhombic
2.08 signal (<i>g</i> = 2.077, 1.935, 1.880). Both signals
displayed relaxation properties similar to those of [4Fe-4S] clusters
and are consistent with an <i>S</i> = <sup>1</sup>/<sub>2</sub> H-cluster containing a [4Fe-4S]<sub>H</sub><sup>+</sup> subcluster.
These EPR signals were correlated with differences in the CO and CN
ligand modes in the FTIR spectra of H<sub>2</sub>- and NaDT-reduced
samples compared with H<sub>ox</sub>. The results indicate that reduction
of [4Fe-4S]<sub>H</sub> from the 2+ state to the 1+ state occurs during
both catalytic H<sub>2</sub> activation and proton reduction and is
accompanied by structural rearrangements of the diiron subsite CO/CN
ligand field. Changes in the [4Fe-4S]<sub>H</sub> oxidation state
occur in electron exchange with the diiron subsite during catalysis
and mediate electron transfer with either external carriers or accessory
FeS clusters
Paradigm Shift for Radical S-Adenosyl- l -methionine Reactions: The Organometallic Intermediate Ļ Is Central to Catalysis
Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5ā²-deoxyadenosyl radical (5ā²-dAdoĀ·). Our recent observation of a catalytically competent organometallic intermediate Ī© that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ī© in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ī© forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ī© as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ī© involves an FeāC5ā² bond between 5ā²-dAdoĀ· and the [4Feā4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5ā²-dAdoĀ· intermediate. Liberation of a reactive 5ā²-dAdoĀ· intermediate via homolytic metalācarbon bond cleavage thus appears to be similar for Ī© and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (ā¼12) of distinct reactions, whereas the RS superfamily has more than 100āÆ000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ī© across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.ISSN:0002-7863ISSN:1520-512
Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme
Pyruvate formate-lyase
activating enzyme (PFL-AE) is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme that installs
a catalytically essential glycyl radical on pyruvate formate-lyase.
We show that PFL-AE binds a catalytically essential monovalent cation
at its active site, yet another parallel with B<sub>12</sub> enzymes,
and we characterize this cation site by a combination of structural,
biochemical, and spectroscopic approaches. Refinement of the PFL-AE
crystal structure reveals Na<sup>+</sup> as the most likely ion present
in the solved structures, and pulsed electron nuclear double resonance
(ENDOR) demonstrates that the same cation site is occupied by <sup>23</sup>Na in the solution state of the as-isolated enzyme. A SAM
carboxylate-oxygen is an M<sup>+</sup> ligand, and EPR and circular
dichroism spectroscopies reveal that both the site occupancy and the
identity of the cation perturb the electronic properties of the SAM-chelated
ironāsulfur cluster. ENDOR studies of the PFL-AE/[<sup>13</sup>C-methyl]-SAM complex show that the target sulfonium positioning
varies with the cation, while the observation of an isotropic hyperfine
coupling to the cation by ENDOR measurements establishes its intimate,
SAM-mediated interaction with the cluster. This monovalent cation
site controls enzyme activity: (i) PFL-AE in the absence of any simple
monovalent cations has littleāno activity; and (ii) among monocations,
going down Group 1 of the periodic table from Li<sup>+</sup> to Cs<sup>+</sup>, PFL-AE activity sharply maximizes at K<sup>+</sup>, with
NH<sub>4</sub><sup>+</sup> closely matching the efficacy of K<sup>+</sup>. PFL-AE is thus a type I M<sup>+</sup>-activated enzyme whose
M<sup>+</sup> controls reactivity by interactions with the cosubstrate,
SAM, which is bound to the catalytic ironāsulfur cluster