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Dihydroorotate dehydrogenases (DHODs) catalyze the transfer of electrons between dihydroorotate and specific oxidant substrates. Class 1B DHODs (DHODBs) use NAD+ as the oxidant substrate and have a heterodimeric structure that incorporates two active sites, each with a flavin cofactor. One Fe2S2 center lies roughly equidistant between the flavin isoalloxazine rings. This arrangement allows for simultaneous association of reductant and oxidant substrates. Here we describe a series of experiments designed to reveal sequences and contingencies in DHODB chemistry. From these data it was concluded that the resting state of the enzyme is FAD•Fe2S2•FMN. Reduction by either NADH or DHO results in two electrons residing on the FMN cofactor that has a 47 mV higher reduction potential than the FAD. The FAD•Fe2S2•FMNH2 state accumulates with a bisemiquinone state that is an equilibrium accumulation formed from a partial transfer of one electron to the FAD. Pyrimidine reduction is reliant on the availability of the Cys135 proton, as the C135S variant slows orotate reduction by ∼40-fold. The rate of pyrimidine reduction is modulated by occupancy of the FAD site; NADH•FAD•Fe2S2•FMNH2•orotate complex can reduce the pyrimidine at 16 s–1, while NAD+•FAD•Fe2S2•FMNH2•orotate complex reduces the pyrimidine at 5.4 s–1 and the FAD•Fe2S2•FMNH2•orotate complex at 0.6 s–1. This set of effector states account for the apparent discrepancy in the slowest rate observed in transient state single turnover reactions with limiting NADH and the limiting rate observed in steady state.
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Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy
NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV0) of the opening transition. While the ΔV0 characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.
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- PAR ID:
- 10354006
- Date Published:
- Journal Name:
- Biology
- Volume:
- 11
- Issue:
- 4
- ISSN:
- 2079-7737
- Page Range / eLocation ID:
- 510
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/biology11040510
