Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase

SummaryNADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this

Role of transmembrane pH gradient and membrane binding in nisin pore formation

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential (Delta psi) and that involves local pertubation of the lipid bilayer structure. Here me show that nisin does not form exclusively voltage dependent pores: even in the absence of a Delta psi, nisin is able to dissipate the transmembrane pH gradient (Delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the Delta pH. Nisin forms pores only when the Delta pH is inside alkaline. The efficiency of Delta psi-induced pore formation is strongly affected by the external pH, whereas Delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-Delta Ala(5))-nisin(1-32) amide have a strongly reduced capacity to dissipate the Delta psi and Delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells, Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding, Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation (A, J. M. Driessen et al., Biochemistry 34:1606-1614, 1995).</p

Solution structure of the cytochrome P450 reductase–cytochrome c complex determined by neutron scattering

Electron transfer in all living organisms critically relies on formation of complexes between the proteins involved. The function of these complexes requires specificity of the interaction to allow for selective electron transfer but also a fast turnover of the complex, and they are therefore often transient in nature, making them challenging to study. Here, using small-angle neutron scattering with contrast matching with deuterated protein, we report the solution structure of the electron transfer complex between cytochrome P450 reductase (CPR) and its electron transfer partner cytochrome c This is the first reported solution structure of a complex between CPR and an electron transfer partner. The structure shows that the interprotein interface includes residues from both the FMN- and FAD-binding domains of CPR. In addition, the FMN is close to the heme of cytochrome c but distant from the FAD, indicating that domain movement is required between the electron transfer steps in the catalytic cycle of CPR. In summary, our results reveal key details of the CPR catalytic mechanism, including interactions of two domains of the reductase with cytochrome c and motions of these domains relative to one another. These findings shed light on interprotein electron transfer in this system and illustrate a powerful approach for studying solution structures of protein-protein complexes

Timing and pace of ice-sheet withdrawal across the marine-terrestrial transition west of Ireland during the last glaciation

Understanding the pace and drivers of marine-based ice-sheet retreat relies upon the integration of numerical ice-sheet models with observations from contemporary polar ice sheets and well-constrained palaeo-glaciological reconstructions. This paper provides a reconstruction of the retreat of the last British–Irish Ice Sheet (BIIS) from the Atlantic shelf west of Ireland during and following the Last Glacial Maximum (LGM). It uses marine-geophysical data and sediment cores dated by radiocarbon, combined with terrestrial cosmogenic nuclide and optically stimulated luminescence dating of onshore ice-marginal landforms, to reconstruct the timing and rate of ice-sheet retreat from the continental shelf and across the adjoining coastline of Ireland, thus including the switch from a marine- to a terrestrially-based ice-sheet margin. Seafloor bathymetric data in the form of moraines and grounding-zone wedges on the continental shelf record an extensive ice sheet west of Ireland during the LGM which advanced to the outer shelf. This interpretation is supported by the presence of dated subglacial tills and overridden glacimarine sediments from across the Porcupine Bank, a westwards extension of the Irish continental shelf. The ice sheet was grounded on the outer shelf at ~26.8 ka cal bp with initial retreat underway by 25.9 ka cal bp. Retreat was not a continuous process but was punctuated by marginal oscillations until ~24.3 ka cal bp. The ice sheet thereafter retreated to the mid-shelf where it formed a large grounding-zone complex at ~23.7 ka cal bp. This retreat occurred in a glacimarine environment. The Aran Islands on the inner continental shelf were ice-free by ~19.5 ka bp and the ice sheet had become largely terrestrially based by 17.3 ka bp. This suggests that the Aran Islands acted to stabilize and slow overall ice-sheet retreat once the BIIS margin had reached the inner shelf. Our results constrain the timing of initial retreat of the BIIS from the outer shelf west of Ireland to the period of minimum global eustatic sea level. Initial retreat was driven, at least in part, by glacio-isostatically induced, high relative sea level. Net rates of ice-sheet retreat across the shelf were slow (62–19 m a−1) and reduced (8 m a−1) as the ice sheet vacated the inner shelf and moved onshore. A picture therefore emerges of an extensive BIIS on the Atlantic shelf west of Ireland, in which early, oscillatory retreat was followed by slow episodic retreat which decelerated further as the ice margin became terrestrially based. More broadly, this demonstrates the importance of localized controls, in particular bed topography, on modulating the retreat of marine-based sectors of ice sheets

Fluctuations, dissipation and the dynamical Casimir effect

Vacuum fluctuations provide a fundamental source of dissipation for systems coupled to quantum fields by radiation pressure. In the dynamical Casimir effect, accelerating neutral bodies in free space give rise to the emission of real photons while experiencing a damping force which plays the role of a radiation reaction force. Analog models where non-stationary conditions for the electromagnetic field simulate the presence of moving plates are currently under experimental investigation. A dissipative force might also appear in the case of uniform relative motion between two bodies, thus leading to a new kind of friction mechanism without mechanical contact. In this paper, we review recent advances on the dynamical Casimir and non-contact friction effects, highlighting their common physical origin.Comment: 39 pages, 4 figures. Review paper to appear in Lecture Notes in Physics, Volume on Casimir Physics, edited by Diego Dalvit, Peter Milonni, David Roberts, and Felipe da Rosa. Minor changes, a reference adde