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There is a mounting body of evidence to suggest that enzyme motions are linked to function, although the design of informative experiments aiming to evaluate how this motion facilitates reaction chemistry is challenging. For the family of diflavin reductase enzymes, typified by cytochrome P450 reductase, accumulating evidence suggests that electron transfer is somehow coupled to large-scale conformational change and that protein motions gate the electron transfer chemistry. These ideas have emerged from a variety of experimental approaches, including structural biology methods (i.e. X-ray crystallography, electron paramagnetic/NMR spectroscopies and solution X-ray scattering) and advanced spectroscopic techniques that have employed the use of variable pressure kinetic methodologies, together with solvent perturbation studies (i.e. ionic strength, deuteration and viscosity). Here, we offer a personal perspective on the importance of motions to electron transfer in the cytochrome P450 reductase family of enzymes, drawing on the detailed insight that can be obtained by combining these multiple structural and biophysical approaches. [ABSTRACT FROM AUTHOR]

Abstract: Biological electron transfer is a fundamentally important reaction. Despite the apparent simplicity of these reactions (in that no bonds are made or broken), their experimental interrogation is often complicated because of adiabatic control exerted through associated chemical and conformational change. We have studied the nature of this control in several enzyme systems, cytochrome P450 reductase, methionine synthase reductase and copper-dependent nitrite reductase. Specifically, we review the evidence for conformational control in cytochrome P450 reductase and methionine synthase reductase and chemical control i.e. proton coupled electron transfer in nitrite reductase. This evidence has accrued through the use and integration of structural, spectroscopic and advanced kinetic methods. This integrated approach is shown to be powerful in dissecting control mechanisms for biological electron transfer and will likely find widespread application in the study of related biological redox systems. [Copyright &y& Elsevier]

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]

This study on human cytochrome P450 reductase (CPR) presents a comprehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. pH-dependent redox potentiometry revealed that the thermodynamic equilibrium between various two-electron reduced enzyme species (FMNH•,FADH•; FMN,FADH2; FMNH2,FAD) is independent of pH. No shift from the blue, neutral di-semiquinone (FMNH•,FADH•) towards the red, anionic species is observed upon increasing the pH from 6.5 to 8.5. Spectrophotometric analysis of events following the mixing of oxidized CPR and NADPH (1 to 1) in a stopped-flow instrument demonstrates that the establishment of this thermodynamic equilibrium becomes a very slow process at elevated pH, indicative of a pH-gating mechanism. The final level of blue di-semiquinone formation is found to be pH independent. Stopped-flow experiments using excess NADPH over CPR provide evidence that both pH and solvent significantly influence the kinetic exposure of the blue di-semiquinone intermediate, yet the observed rate constants are essentially pH independent. Thus, the kinetic pH-gating mechanism under stoichiometric conditions is of no significant kinetic relevance for four-electron reduction, but rather modulates the observed semiquinone absorbance at 600 nm in a pH-dependent manner. The use of proton inventory experiments and primary kinetic isotope effects are described as kinetic tools to disentangle the intricate pH-dependent kinetic mechanism in CPR. Our analysis of the pH and isotope dependence in human CPR reveals previously hidden complexity in the mechanism of electron transfer in this complex flavoprotein. [ABSTRACT FROM AUTHOR]

The central photochemical reaction in photosystem II of green algae and plants and the reaction center of some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule. Through protein engineering, we have been able to modify a protein to mimic this reaction. A unique quinonesbinding site was engineered into the Estherkhia coil cytochrome b562 by introducing a cysteine within the hydrophobic interior of the protein Various quinones, such as p-benzoquinone and 2,3-dimethoxy-5-methyl-1 ,4-benzoquinone, were then covalently attached to the protein through a cysteine sulfur addition reaction to the quinone ring. The cysteine placement was designed to bind the quinone ∼10 Å from the edge of the bound porphyrin. Fluorescence measurements confirmed that the bound hydroquinone is incorporated toward the protein's hydrophobic interior and is partially solvent-shielded. The bound quinones remain redox-active and can be oxidized and rereduced in a two-electron process at neutral pH. The semiquinone can be generated at high pH by a one-electron reduction, and the mid- point potential of this can be adjusted by ∼500 my by binding different quinones to the protein. The heme-binding site of the modified cytochrome was then reconstituted with the chlorophyll analogue zinc chlorin e6. By using EPR and fast optical techniques, we show that in the various chlorin-protein-quinone complexes, light-induced electron transfer can occur from the chlorin to the bound oxidized quinone but not the hydroquinone, with electron transfer rates in the order of 108 s-1. [ABSTRACT FROM AUTHOR]

The reduction of nitrite (NO2-) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H2O and D2O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ∼1.33 ± 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substratefree AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H2O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mM. As a consequence, the observed ET process was faster in D2o than in H2O above 0.1 mM nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ∼0.61 ± 0.02 between 3 and 10 mM. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover. [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]

This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue α3Y three-helix bundle. α3Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from α3Y at both acidic (pH 5.6) and alkaline (pH 8.3) conditions. The observed Faradaic response is uniquely associated with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton-coupled electron-transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold. α3Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. α3Y-K29H and α3Y-K36H each contain a histidine residue whose protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical content, tertiary structure, solution aggregation state, and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3-4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects. [ABSTRACT FROM AUTHOR]