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Research has been conducted on the phylloquinone secondary electron acceptor A (sub)1. The environment of this acceptor within the photosystem I reaction center has been investigated via site-directed mutagenesis of tryptophan residues in the Chlamydomonas reinhardtii PsaA sumunit.

Oxidative stress plays a key role in Alzheimer's disease (AD). In addition, the abnormally high Cu[sup2+] ion concentrations present in senile plaques has provoked a substantial interest in the relationship between the amyloid β peptide (Aβ) found within plaques and redox-active copper ions. There have been a number of studies monitoring reactive oxygen species (ROS) generation by copper and ascorbate that suggest that Aβ acts as a prooxidant producing H[sub2[O[sub2]. However, others have indicated Aβ acts as an antioxidant, but to date most cell-free studies directly monitoring ROS have not supported this hypothesis. We therefore chose to look again at ROS generation by both monomeric and fibrillar forms of Aβ under aerobic conditions in the presence of Cu[sup2+] with/Without the biological reductant ascorbate in a cell-free system. We used a variety of fluorescence and absorption based assays to monitor the production of ROS, as well as Cu[sup2+] reduction. In contrast to previous studies, we show here that Aβ does not generate any more ROS than controls of Cu[sup2+] and ascorbate. Aβ does not silence the redox activity of Cu[sup2+] via chelation, but rather hydroxyl radicals produced as a result of Fenton-Haber Weiss reactions of ascorbate and Cu[sup2+] rapidly react with Aβ; thus the potentially harmful radicals are quenched. In support of this, chemical modification of the Aβ peptide was examined using [sup1]H NMR, and specific oxidation sites within the peptide were identified at the histidine and methionine residues. Our studies add significant weight to a modified amyloid cascade hypothesis in which sporadic AD is the result of Aβ being upregulated as a response to oxidative stress. However, our results do not preclude the possibility that Aβ in an oligomeric form may concentrate the redox-active copper at neuronal membranes and so cause lipid peroxidation. [ABSTRACT FROM AUTHOR]

Multiple solution-state techniques have been employed in investigating the nature and control of electron transfer in the context of the proposed ‘domain shuffle hypothesis’ for intraprotein electron transfer inferred from the crystal structure of the nitric oxide synthase reductase domain. NADPH analogues and fragments have been used to map those regions of this substrate that are important in eliciting a conformational change, observed in both the fluorescence emission of the flavin cofactors of the enzyme and the EPR spectra of the FMN flavosemiquinone state. EPR and UV-visible potentiometric methods have demonstrated a substantial calmodulin-dependent perturbation in the midpoint reduction potentials of the redox couples of both flavin cofactors, in contrast to a previous report [Noble, M. A., et al. (1999) Biochemistry 38, 16413–16418]. These studies support a model in which FMN domain mobility, triggered by Ca2+-calmodulin binding and antagonized by substrate binding, facilitates electron transfer in nitric oxide synthase through conformational change and effects a major change in the midpoint reduction potentials of the flavin redox couples. These results are discussed in light of the recent crystal structure of the NADPH-locked reductase domain. [ABSTRACT FROM AUTHOR]

Dihydroorotate dehydrogenase B (DHODB) is a complex iron--sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD+. The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 ± 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E1) and semiquinone/ hydroquinone (E2) couples for the FMN (E1 = -301 ± 6 mV; E2 = -252 ± 8 mV) and FAD (E1 = -312 ± 6 mV; E2 = -297 ± 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E1 = -298 ± 4 mV; E2 = -259 ± 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at ∼630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein. [ABSTRACT FROM AUTHOR]

Discusses the study of cation radicals of the primary donor, P680, and two secondary donor chlorophylls in photosystem 2 (PS2) using electron nuclear double resonance (ENDOR) and special triple (ST) resonance spectroscopes. Description of materials and methods; Results of study; Discussion of results; References.

Conformational control limits most electron transfer (ET) reactions in biology, but we lack general insight into the extent of conformational space explored, and specifically the properties of the associated energy landscape. Here we unite electron-electron double resonance (ELDOR) studies of the diradical (disemiquinoid) form of human cytochrome P450 reductase (CPR), a nicotinamide adenine phosphate dinucleotide (NADPH)-linked diflavin oxidoreductase required for P450 enzyme reduction, with functional studies of internal ET to gain new insight into the extent and properties of the energy landscape for conformationally controlled ET. We have identified multiple conformations of disemiquinoid CPR, which point to a rugged energy landscape for conformational sampling consistent with functional analysis of ET using high-pressure stopped-flow, solvent, and temperature perturbation studies. Crystal structures of CPR have identified discrete "closed" and "open" states, but we emphasize the importance of a continuum of conformational states across the energy landscape. Within the landscape more closed states that favor internal ET are formed by nucleotide binding. Open states that enable P450 enzymes to gain access to electrons located in the FMN-domain are favored in the absence of bound coenzyme. The extent and nature of energy landscapes are therefore accessible through the integration of ELDOR spectroscopy with functional studies. We suggest this is a general approach that can be used to gain new insight into energy landscapes for biological ET mediated by conformational sampling mechanisms. [ABSTRACT FROM AUTHOR]

Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure-activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N-H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of C c P W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in C c P W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cyt c . Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor. This raises the question how efficient decarboxylation of (hetero)aromatic acids is achieved by other UbiD family members. Here, we show that the Pseudomonas aeruginosa virulence attenuation factor PA0254 / HudA is a pyrrole-2-carboxylic acid decarboxylase. The crystal structure of the enzyme in the presence of the reversible inhibitor imidazole reveals a covalent prFMN - imidazole adduct is formed. Substrate screening reveals HudA and selected active site variants can accept a modest range of heteroaromatic compounds, including thiophene-2-carboxylic acid. Together with computational studies, our data suggests prFMN covalent catalysis occurs via electrophilic aromatic substitution and links HudA activity with the inhibitory effects of pyrrole-2-carboxylic acid on P. aeruginosa quorum sensing., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid ( K D = 2.7 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 ( K M = 3.1 μM compared to 330 μM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors ( E 0 ' ∼ -118 mV), and a large increase in the CYP505A30 heme domain Fe III /Fe II redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30's ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications., Competing Interests: The authors declare no competing financial interest.