George T. Gassner

Structure and mechanism of the iron-sulfur flavoprotein phthalate dioxygenase reductase.

Transfer of electrons between pyri‐dine nucleotides (obligatory two‐electron carriers) and hemes or [2Fe‐2S] centers (obligatory one‐electron carriers) is an essential step mediated by flavins in respiration, photosynthesis, and many oxygenase systems. Phthalate dioxygenase reductase (PDR), a soluble iron‐sulfur flavoprotein from Pscudomofias cepacia, is a convenient model for the study of this type of electron transfer. PDR is folded into thrjee domains; the NH2terminal FMN binding and central NAD(H) binding domains are closely related to ferredoxin‐NADP+ reductase (FNR). The COOH‐términal [2Fe‐2S] domain is similar to plaUt ferredoxins, and can be removed by proteolysis without significantly altering the reactivity of the FNR‐like domains. Kinetic studies have identified sequential steps in the reaction of PDR with NADH that involve pyridine nucleotide binding, hydrijde transfer to FMN, and intramolecular electron transfer from the reduced flavin to the [2Fe‐2S] cluster. Crystal structures of reduced and liganded PDR correspond to some of the intermediates formed during reduction by NADH. Small structural changes that are observed in the vicinity of the cofactors upon reduction or NAD(H) binding may provide part of the reorganization energy or contribute to the gating mechanism that controls intramolecular electron transfer.—Gassner, G. T., Ludwig, M. L., Gatti, D. L., Correll, C. C., Ballou, D. P. Structure and mechanism of the iron‐sulfur flavoprotein phthalate dioxygenase reductase. FASEB J. 9, 1411‐1418 (1995)

Nature of the reaction intermediates in the flavin adenine dinucleotide-dependent epoxidation mechanism of styrene monooxygenase.

Styrene monooxygenase (SMO) is a two-component flavoenzyme composed of an NADH-specific flavin reductase (SMOB) and FAD-specific styrene epoxidase (NSMOA). NSMOA binds tightly to reduced FAD and catalyzes the stereospecific addition of one atom of molecular oxygen to the vinyl side chain of styrene in the enantioselective synthesis of S-styrene oxide. In this mechanism, molecular oxygen first reacts with NSMOA(FAD(red)) to yield an FAD C(4a)-peroxide intermediate. This species is nonfluorescent and has an absorbance maximum of 382 nm. Styrene then reacts with the peroxide intermediate with a second-order rate constant of (2.6 ± 0.1) × 10(6) M(-1) s(-1) to yield a fluorescent intermediate with an absorbance maximum of 368 nm. We compute an activation free energy of 8.7 kcal/mol for the oxygenation step, in good agreement with that expected for a peroxide-catalyzed epoxidation, and acid-quenched samples recovered at defined time points in the single-turnover reaction indicate that styrene oxide synthesis is coincident with the formation phase of the fluorescent intermediate. These findings support FAD C(4a)-peroxide being the oxygen atom donor and the identity of the fluorescent intermediate as an FAD C(4a)-hydroxide product of the styrene epoxidation. Overall, four pH-dependent rate constants corresponding to peroxyflavin formation (pK(a) = 7.2), styrene epoxidation (pK(a) = 7.7), styrene oxide dissociation (pK(a) = 8.3), and hydroxyflavin dehydration (pK(a) = 7.6) are needed to fit the single-turnover kinetics.

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 → FADox 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 FADhq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq 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 FADhq to NSMOA. The binding of pyridine nucleotides to SMOB-FADhq shifts the FADhq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB-FADox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions.

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

Abstract Water proton T1–1 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.