Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochromes P450 via its FAD and FMN.To understand the biochemical and structural basis of electron transfer from FMN-hydroquinone to its partners, three deletion mutants in a conserved loop near the FMN were characterized. Comparison of oxidized and reduced wild type and mutant structures reveals that the basis for the air stability of the neutral blue semiquinone is protonation of the flavin N5 and strong H-bond formation with the Gly-141 carbonyl. The ⌬Gly-143 protein had moderately decreased activity with cytochrome P450 and cytochrome c. It formed a flexible loop, which transiently interacts with the flavin N5, resulting in the generation of both an unstable neutral blue semiquinone and hydroquinone. The ⌬Gly-141 and ⌬G141/E142N mutants were inactive with cytochrome P450 but fully active in reducing cytochrome c. In the ⌬Gly-141 mutants, the backbone amide of Glu/Asn-142 forms an H-bond to the N5 of the oxidized flavin, which leads to formation of an unstable red anionic semiquinone with a more negative potential than the hydroquinone. The semiquinone of ⌬G141/E142N was slightly more stable than that of ⌬Gly-141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both ⌬Gly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the neuronal NOS mutant (⌬Gly-810). Our results indicate that the catalytic activity of cytochrome P450 oxidoreductase is a function of the length, sequence, and flexibility of the 140s loop and illustrate the sophisticated variety of biochemical mechanisms employed in fine-tuning its redox properties and function.

Section: Determination Of the Redox Potentials Of The Fmn Domainssupporting

confidence: 78%

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confidence: 99%

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confidence: 99%

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Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

Rwere

Xia

et al. 2016

“…Presumably this prevents the required conformational change and/or H-bond stabilization of the neutral sq radical. Validation of this model comes from elegant experiments in which the relative values of E 1 and E 2 are reversed, either by deleting the loop Gly in NOS (45) or by inserting the loop Gly in P450-BM3 (46).…”

Section: Discussionmentioning

confidence: 99%

Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

Damiani

Nostedt

O’Neill

2011

Flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. A versatile regulatory tool is proton transfer. Here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (PLs) and cryptochromes (CRYs). These light-responsive proteins share homologous overall architectures and FAD-binding pockets, yet they have evolved divergent functions that include DNA repair, photomorphogenesis, regulation of circadian rhythm, and magnetoreception. We report the first measurement of both FAD redox potentials for cyclobutane pyrimidine dimer PL (CPD-PL, Anacystis nidulans). These values, E 1 (hq/sq) ‫؍‬ ؊140 mV and E 2 (sq/ox) ‫؍‬ ؊219 mV, where hq is FAD hydroquinone and ox is oxidized FAD, establish that the sq is not thermodynamically stabilized (⌬E ‫؍‬ E 2 ؊ E 1 ‫؍‬ ؊79 mV). Results with N386D CPD-PL support our earlier hypothesis of a kinetic barrier to sq oxidation associated with proton transfer. Both E 1 and E 2 are upshifted by ϳ100 mV in this mutant; replacing the N5-proximal Asn with Asp decreases the driving force for sq oxidation. However, this Asp alleviates the kinetic barrier, presumably by acting as a proton shuttle, because the sq in N386D CPD-PL oxidizes orders of magnitude more rapidly than wild type. These data clearly reveal, as suggested for plant CRYs, that an N5-proximal Asp can switch on proton transfer and modulate sq reactivity. However, the effect is context-dependent. More generally, we propose that PLs and CRYs tune the properties of their N5-proximal residue to adjust the extent of proton transfer, H-bonding patterns, and changes in protein conformation associated with electron transfer at the flavin. Electron transfer (ET)2 within and between proteins is a ubiquitous and essential molecular process in biology. Proteins must tightly control the rates of ET and lifetimes of radical intermediates to use this fundamental chemical reaction for a vast array of cellular functions. A significant control mechanism involves coupling of the ET to a proton transfer (PT) (1-3). The PT ensures heightened discrimination toward subtle changes in protein structure. As compared with ET, it has a much shorter range and is very sensitive to the relative orientation of a few atoms (the proton, donor, and acceptor), and its kinetics and thermodynamics can be adjusted dramatically by tuning pK a values over a very large range (3). Consequently, proton-coupled ET (PCET) is widespread in nature, notably in biological energy conversion and redox catalysis. Understanding the mechanisms for PCET and identifying its role in protein evolution are central problems in chemical biology.Flavoproteins are major players in cellular redox reactions (4). The flavin cofactor can exist in three redox states: the two-electron reduced hydroquinone (hq), which is often anionic; the one-electron reduced semiquinone (sq), as a neutral or anionic radical; and the fully oxidized form (ox) (Fig. 1a). Flavoprotein...

“…Heme was added as an albumin conjugate during the expression to convert all of the nNOS to the holo-nNOS dimer. The human CaM plasmid, pACYC/ trc-hCaM, was a gift from the R. Neubig laboratory (University of Michigan), and expression and purification were performed essentially as described (27).…”

Section: Methodsmentioning

confidence: 99%

Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

Yokom¹,

Morishima²,

Lau³

et al. 2014

Background: NOS enzymes are large, dimeric complexes essential in mammalian physiology. Results: EM structural analysis and three-dimensional models reveal nNOS reductase-oxygenase arrangements and a CaMdependent rotation of the FMN domain. Conclusion: Coordinated conformational changes act to reposition the FMN domain for electron transfer. Significance: This work captures structural states of the NOS holoenzyme that drive the NO synthesis cycle.