Structure and mechanism of the iron‐sulfur flavoprotein phthalate dioxygenase reductase

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154520/1/fsb2009014006.pd

NMRD studies on phthalate dioxygenase: evidence for displacement of water on binding substrate

 Water proton T 1 –1 measurements at magnetic fields between 0.01 and 50 MHz [nuclear magnetic relaxation dispersion (NMRD) measurements] have been performed on solutions of phthalate dioxygenase (PDO) reconstituted at the catalytic iron site with copper(II) or manganese(II). The data show evidence of a weakly coordinated water molecule in CuPDO; in the presence of the substrate, phthalate, this water appears to become even less tightly bound, and an additional tightly coordinated water can be detected. In PDO reconstituted with manganese, one tightly coordinated water is detected in the presence and in the absence of phthalate. An attempt is made to reconcile these data with low-temperature near-IR magnetic circular dichroism and X-ray absorption data, which show that PDO reconstituted with iron or cobalt is six-coordinate in the absence of substrate and five-coordinate in the presence of substrate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42322/1/775-1-5-468_60010468.pd

Carbon Dioxide Utilisation -The Formate Route

UIDB/50006/2020 CEEC-Individual 2017 Program Contract.The relentless rise of atmospheric CO2 is causing large and unpredictable impacts on the Earth climate, due to the CO2 significant greenhouse effect, besides being responsible for the ocean acidification, with consequent huge impacts in our daily lives and in all forms of life. To stop spiral of destruction, we must actively reduce the CO2 emissions and develop new and more efficient “CO2 sinks”. We should be focused on the opportunities provided by exploiting this novel and huge carbon feedstock to produce de novo fuels and added-value compounds. The conversion of CO2 into formate offers key advantages for carbon recycling, and formate dehydrogenase (FDH) enzymes are at the centre of intense research, due to the “green” advantages the bioconversion can offer, namely substrate and product selectivity and specificity, in reactions run at ambient temperature and pressure and neutral pH. In this chapter, we describe the remarkable recent progress towards efficient and selective FDH-catalysed CO2 reduction to formate. We focus on the enzymes, discussing their structure and mechanism of action. Selected promising studies and successful proof of concepts of FDH-dependent CO2 reduction to formate and beyond are discussed, to highlight the power of FDHs and the challenges this CO2 bioconversion still faces.publishersversionpublishe

Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD-Transfer Reaction

The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FAD_ox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s^–1, in a step that is coupled to the rapid dissociation of NAD⁺. Electrochemical and equilibrium-binding studies indicate that NSMOA binds FAD_hq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FAD_hq from the reductase to the epoxidase. After binding to NSMOA, FAD_hq rapidly reacts with molecular oxygen to form a stable C­(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein–protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD⁺ after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FAD_hq to NSMOA. The binding of pyridine nucleotides to SMOB–FAD_hq shifts the FAD_hq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB–FAD_ox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions