Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains

Abstract

How the dynamics of proteins assist catalysis is a contemporary issue in enzymology. In particular, this holds true for membrane-bound enzymes, where multiple structural, spectroscopic and biochemical approaches are needed to build up a comprehensive picture of how dynamics influence enzyme reaction cycles. Of note are the recent studies of cytochrome P450 reductases (CPR)-P450 (CYP) endoplasmic reticulum redox chains, showing the relationship between dynamics and electron flow through flavin and haem redox centres and the impact this has on monooxygenation chemistry. These studies have led to deeper understanding of mechanisms of electron flow, including the timing and control of electron delivery to protein-bound cofactors needed to facilitate CYP-catalysed reactions. Individual and multiple component systems have been used to capture biochemical behaviour and these have led to the emergence of more integrated models of catalysis. Crucially, the effects of membrane environment and composition on reaction cycle chemistry have also been probed, including effects on coenzyme binding/release, thermodynamic control of electron transfer, conformational coupling between partner proteins and vectorial versus 'off pathway' electron flow. Here, we review these studies and discuss evidence for the emergence of dynamic structural models of electron flow along human microsomal CPR-P450 redox chains.

Keywords: cytochrome P450; cytochrome P450 reductase; electron transfer chemistry; membrane protein; protein domain dynamics.

Figure 1

Cellular location and function of mammalian CPR. CPR is anchored to the cytosolic side of the ER, where it receives electrons from NADPH before passing them to a variety of partner proteins. The partner proteins of CPR include HO (shown as a red sphere), squalene monooxygenase (SQLE, shown as a yellow sphere), cytochrome b ₅ (cyt b ₅, shown as a brown sphere) and the superfamily of cytochrome P450 proteins (CYPs, shown as an orange sphere). CPR is also known to activate a wide variety of prodrugs, such as the anticancer drug, mitomycin C.

Figure 2

X‐ray structures of (A) ‘closed’ CPR, (PDB ID 1AMO), (B) ‘open’ (ΔTGEE) CPR (PDB ID 3ES9) and (C) HO‐bound ‘open’ (ΔTGEE) CPR (PDB ID 3WKT). The FAD‐containing, connecting, and FMN‐containing domains of CPR are shown as dark blue, marine blue and light blue cartoons respectively. HO is shown as a red cartoon. The FAD, FMN, NADP⁺ and haem cofactors are shown as dark blue, cyan, yellow and red respectively.

Figure 3

The reaction mechanism of CPR. (A) shows an example stopped‐flow transient for CPR flavin reduction. The CPR reductive half reaction comprises three kinetic phases. The first two of these kinetic phases are loosely related to two‐ and four‐electron reduction, while the third, slow phase is related to the EQ state of CPR. This EQ state is hypothesized to be a conformational change and/or further oxidation of NADPH attributed to thermodynamic relaxation. (B) shows the proposed mechanism of CPR catalysed flavin reduction. In (B) the QE state refers to the quasi‐equilibrium state of CPR, a state where electrons are distributed between the FAD and FMN cofactors.

Figure 4

Correlating dynamics with the enzyme reaction cycle using ‘real‐time’ methods. (A) A schematic showing how RAS has been used to probe ‘real‐time’ dynamics of CPR related to redox state. (B) A schematic showing how CPR domain dynamics have been linked to the reaction coordinate through the use of FRET stopped‐flow methods. As the three‐electron reduced, ‘open’ form of CPR has not been investigated/detected by stopped‐flow FRET methods, we have omitted the fluorophores bound from this CPR conformation in (B).

Figure 5

The relationship between CPR dynamics and the enzyme reaction cycle. The two models that have been proposed for CPR dynamics linked to catalysis. In the model presented at the top, CPR moves from a more ‘open’ state to a ‘closed’ state when reduced with one equivalent of NADPH. Following reduction by a second NADPH equivalent, CPR is predominantly in an ‘open’ state compatible with electron transfer to partner proteins. In the second model (below), the enzyme is ‘closed’ in the oxidized, coenzyme‐free state. Upon reduction with NADPH, CPR opens and electrons can be passed to partner proteins. It should be noted that, as CPR is purified in a one‐electron reduced state, it is often hypothesized that CPR may cycle between one‐ and three‐electron reduced states in vivo and not the two‐ and four‐electron reduced states portrayed here.