Laura S. Busenlehner

Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase

Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of O2 to H2O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a “gate,” which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molecular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogen/deuterium exchange MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the framework of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation.

Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics

Background: SufS cysteine desulfurase mobilizes sulfur for stress-responsive iron-sulfur cluster biogenesis in bacteria. Results: Interaction with the sulfur transfer protein SufE triggers conformational changes in the SufS active site. Conclusion: SufE participates in allosteric regulation of SufS activity in addition to being a sulfur acceptor. Significance: New insight into sulfur mobilization and transfer during iron-sulfur cluster metallocofactor assembly is provided. Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from l-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.

The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

Rotenone is a widely used pesticide that induces Parkinsons disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

Probing Backbone Dynamics with Hydrogen/Deuterium Exchange Mass Spectrometry

Protein dynamics can be probed by the solution technique amide hydrogen/deuterium exchange. The exchange rate of hydrogen for deuterium along a peptide backbone is dependent on the extent of hydrogen bonding from secondary structure, accessibility by D2O, and protein motions. Both global and local conformational changes that alter bonding or structure will lead to changes in the amount of deuterium incorporated. The deuterium can be localized via pepsin digestion of the protein and quantified by electrospray ionization mass spectrometry through the mass shifts of the resulting peptides. The technique is emerging as an essential tool to study protein structure in solution due to the exceptional capability of examining both dynamic and structural changes related to protein function.

SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase

Many essential metalloproteins require iron-sulfur (Fe-S) cluster cofactors for their function. In vivo persulfide formation from l-cysteine is a key step in the biogenesis of Fe-S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from l-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe-S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein-protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.