CryoEM structures reveal how the bacterial flagellum rotates and switches direction.

Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or 'switch', limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0 Å) and isolated C-ring in a clockwise pose alone (4.6 Å) and bound to a regulator (5.9 Å). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation.

Kinetic Evidence for an Induced Fit Mechanism in the Binding of the Substrate Camphor by Cytochrome P450cam.

Bacterial cytochrome P450 (P450) 101A1 (P450cam) has served as a prototype among the P450 enzymes and has high catalytic activity towards its cognate substrate, camphor. X-ray crystallography and NMR and IR spectroscopy have demonstrated the existence of multiple conformations of many P450s, including P450cam. Kinetic studies have indicated that substrate binding to several P450s is dominated by a conformational selection process, in which the substrate binds an individual conformer(s) of the unliganded enzyme. P450cam was found to differ in that binding of the substrate camphor is dominated by an induced fit mechanism, in which the enzyme binds camphor and then changes conformation, as evidenced by the equivalence of binding eigenvalues observed when varying both camphor and P450cam concentrations. The accessory protein putidaredoxin had no effect on substrate binding. Estimation of the rate of dissociation of the P450cam·camphor complex (15 s-1) and fitting of the data yield a minimal kinetic mechanism in which camphor binds (1.5 × 107 M-1 s-1) and the initial P450cam•camphor complex undergoes a reversible equilibrium (k forward 112 s-1, k reverse 28 s-1) to a final complex. This induced fit mechanism differs from those reported for several mammalian P450s and bacterial P450BM-3, indicative of the diversity of how P450s recognize multiple substrates. However, similar behavior was not observed with the alternate substrates (+)-α-pinene and 2-adamantanone, which probably utilize a conformational selection process.

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes.

Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6-200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 µM Adx, and higher concentrations were not inhibitory up to at least 50 µM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.