1,157 research outputs found
Great metalloclusters in enzymology
Metallocluster-containing enzymes catalyze some of the most basic redox transformations in the biosphere. The reactions catalyzed by these enzymes typically involve small molecules such as N2, CO, and H2 that are used to generate both chemical building blocks and energy for metabolic purposes. During the past decade, structures have been established for the iron-sulfur-based metalloclusters present in the molybdenum nitrogenase, the iron-only hydrogenase, and the nickel-carbon monoxide dehydrogenase, and for the copper-sulfide-based cluster in nitrous oxide reductase. Although these clusters are built from interactions observed in simpler metalloproteins, they contain novel features that may be relevant for their catalytic function. The mechanisms of metallocluster-containing enzymes are still poorly defined, and represent substantial and continuing challenges to biochemists, biophysicists, and synthetic chemists. These proteins also provide a window into the union of the biological and inorganic worlds that may have been relevant to the early evolution of biochemical catalysis
How many metals does it take to fix N2? A mechanistic overview of biological nitrogen fixation
During the process of biological nitrogen fixation, the enzyme nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein; the Fe protein mediates the coupling of ATP hydrolysis to interprotein electron transfer, whereas the active site of the MoFe protein contains the polynuclear FeMo cofactor, a species composed of seven iron atoms, one molybdenum atom, nine sulfur atoms, an interstitial light atom, and one homocitrate molecule. This Perspective provides an overview of biological nitrogen fixation and introduces three contributions to this special feature that address central aspects of the mechanism and assembly of nitrogenase
Nitrogenase: A nucleotide-dependent molecular switch
In the simplest terms, the biological nitrogen cycle is the reduction of atmospheric dinitrogen (N2) to ammonia with the subsequent reoxidation ammonia to dinitrogen (1). At the reduction level of ammonia, nitrogen incorporated into precursors for biological macromolecules such as proteins and nucleic acids. Reoxidation of ammonia to dinitrogen ("denitrification") by a variety of microbes (by way of nitrite and other oxidation levels of nitrogen) leads to the depletion of the "fixed," biologically usable, nitrogen pool. Besides the relatively small contribution from commercial ammonical fertilizer production, replenishing of the nitrogen pool falls mainly to a limited number of physiologically diverse microbes (e.g. eubacteria and archaebacteria; free-living and symbiotic; aerobic and anaerobic) that contain the nitrogenase enzyme system
Crystallographic Analyses of Ion Channels: Lessons and Challenges
Membrane proteins fascinate at many levels, from their central functional roles in transport, energy transduction, and signal transduction processes to structural questions concerning how they fold and operate in the exotic environments of the membrane bilayer and the water-bilayer interface and to methodological issues associated with studying membrane proteins either in situ or extracted from the membrane. This interplay is beautifully exemplified by ion channels, a collection of integral membrane proteins that mediate the transmembrane passage of ions down their electrochemical potential gradient (for general reviews, see Refs. 1 and 2). Ion channels are key elements of signaling and sensing pathways, including nerve cell conduction, hormone response, and mechanosensation. The characteristic properties of ion channels reflect their conductance, ion selectivity, and gating. Ion channels are often specific for a particular type of ion (such as potassium or chloride) or a class of ions (such as anions) and are typically regulated by conformational switching of the protein structure between "open" and "closed" states. This conformational switching may be gated in response to changes in membrane potential, ligand binding, or application of mechanical forces. Detailed functional characterizations of channels and their gating mechanisms have been achieved, reflecting exquisite methodological advances such as patch clamp methods that can monitor the activities of individual channels (3). Until recently, corresponding information about the three-dimensional structures of channels was not available, reflecting difficulties in obtaining sufficient quantities of membrane proteins for crystallization trials. Happily, this situation has started to change with the structure determinations of the Streptomyces lividans K+ channel (KcsA (4)) and the Mycobacterium tuberculosis mechanosensitive channel (MscL (5)).
A variety of reviews (6-12) have appeared recently that discuss functional implications of these channel structures. This review discusses these developments from a complementary perspective, by considering the implications of these structures from within the larger framework of membrane protein structure and function. Because of space restrictions, this review necessarily emphasizes membrane proteins that are composed primarily of alpha-helical bundles, such as KcsA and MscL, rather than beta-barrel proteins, such as porins, typically found in bacterial outer membranes
Powering brain power: GLUT1 and the era of structure based human transporter biology
Every student of biochemistry quickly appreciates
the central role of glycolysis
in cellular metabolism. What is not usually
addressed in an introductory course
is how glucose gets inside a cell in the
first place. Specialized integral membrane
proteins known as transporters are responsible
for glucose uptake; in mammals,
glucose is imported by members of
the GLUT family of which 14 different
varieties have been identified in humans
[1]. GLUT transporters are members of
the major facilitator superfamily of transporters
and catalyze the facilitated uptake
of glucose in the thermodynamically
favored direction. The most widely distributed
version isGLUT1that is responsible
for getting glucose into red blood
cells and across the blood brain barrier,
among many other roles [2]
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The synthesis of novel porphyrin macrocycles for their use as potential non-linear optical materials
This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The potential organic molecular compounds exhibit for the use as nonlinear optical materials, has received a lot of attention recently. Aromatic compounds containing extensive delocalised π-electronic systems are of particular interest. Porphyrins and metalloporphyrins contain an extensive delocalised 7t-electron system and are amongst those compounds of interest. The ability to manipulate the porphyrins peripheral substitution pattern has led to the synthesis of a range of novel porphyrin macrocycles. A series of meso- substituted porphyrin macrocycles ha been synthesised and the non-linear optical properties of these compounds screened. The synthesis of a series of symmetrical meso- substituted porphyrins was achieved by the reactions of various aldehydes with pyrrole or a dipyrromethane. The synthesis of a series of unsymmetrical porphyrins was achieved by the mixed condensation of two benzaldehydes and pyrrole and also by reactions at the periphery of the symmetrical porphyrins.
The Reverse Saturable Absorption properties of the synthesised porphyrins was tested and was exhibited by a number of these compounds. The RSA properties of the compounds were enhanced on the introduction of an unsymmetrical nature to the macrocycle. Also, the effect is enhanced when the ethynyl moiety was introduced to the porphyrins periphery. This is due to the greater overlap between the macrocycle and the peripheral substituents.DERA Malver
Crystal structure of the Escherichia coli transcription termination factor Rho
During the crystal structure analysis of an ATP-binding cassette (ABC) transporter overexpressed in Escherichia coli, a contaminant protein was crystallized. The identity of the contaminant was revealed by mass spectrometry to be the Escherichia coli transcription terminator factor Rho, structures of which had been previously determined in different conformational states. Although Rho was present at only ∼1% of the target protein (a bacterial homolog of the eukaryotic ABC transporter of mitochondria from Novosphingobium aromaticivorans; NaAtm1), it preferentially crystallized in space group C2 as thin plates that diffracted to 3.30 Å resolution. The structure of Rho in this crystal form exhibits a hexameric open-ring staircase conformation with bound ATP; this characteristic structure was also observed on electron-microscopy grids of the NaAtm1 preparation
The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter
The MetNI methionine importer of Escherichia coli, an ATP Binding Cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of D- and L-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of MetNI mechanism has thus far been limited to a series of crystal structures in an inward facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by L-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well
Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site
The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover
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