Mitochondrial cytochrome bc1 complex performs two functions: It is a respiratory multienzyme complex and it recognizes a mitochondrial targeting presequence. Refined crystal structures of the 11-subunit bc1 complex from bovine heart reveal full views of this bifunctional enzyme. The "Rieske" iron-sulfur protein subunit shows significant conformational changes in different crystal forms, suggesting a new electron transport mechanism of the enzyme. The mitochondrial targeting presequence of the "Rieske" protein (subunit 9) is lodged between the two "core" subunits at the matrix side of the complex. These "core" subunits are related to the matrix processing peptidase, and the structure unveils how mitochondrial targeting presequences are recognized.

The histidine-cobalt distance is very long (2.5 A compared with 1.95-2.2 A in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the metal-carbon bond of the cofactor and favour the formation of the initial radical species. The active site is deeply buried, and the only access to it is through a narrow tunnel along the axis of the TIM barrel domain.

When the dynamic properties of many different proteins are plotted as a function of temperature, biphasic behaviour is observed, with a broad transition centred around 220 K. Atomic mean-square displacements from X-ray crystallography and Mössbauer scattering show this behaviour, as do electron transfer rates and dynamic information from inelastic neutron scattering. Molecular dynamics simulations over a range of temperatures also exhibit a transition at about 220 K: high-temperature atomic fluctuations are dominated by anharmonic collective motions of bonded and nonbonded groups of atoms, but below 220 K the predominant dynamic behaviour is harmonic vibration of individual atoms. Here we show by high-resolution X-ray diffraction that crystalline ribonuclease A does not bind substrate or inhibitor at 212 K but will bind either rapidly at 228 K. Once bound at the higher temperature, inhibitor cannot be washed off after the enzyme is cooled to below the transition temperature. These results suggest that enzyme flexibility is required for catalytic function.

The response regulator protein of bacterial chemotaxis, CheY, is representative of a large family of signal transduction proteins that function as phosphorylation-activated switches to regulate the activities of associated effector domains. These regulators catalyze the metal ion-dependent phosphoryl transfer and dephosphorylation reactions that control the effector activities. The crystal structures of Salmonella typhimurium CheY with and without Mg2+ bound at the active site have been determined and refined at 1.8-A resolution. While the overall structures of metal-bound and metal-free CheY are similar, significant rearrangements occur within the active site involving the three most highly conserved residues of the response regulator family. Conservation of the cluster of carboxylate side chains at the active site of response regulator domains can be rationalized in terms of their role in coordinating the catalytically essential divalent metal ion. The Mg2+ coordination geometry provides insights to the mechanism of phosphoryl transfer.