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Cytochrome P450cam (CYP101A1) catalyzes the region- and stereo-specific 5-exo-hydroxylation of camphor via a multistep catalytic cycle that involves two-electron transfer steps, with an absolute requirement that the second electron be donated by the ferrodoxin, putidaredoxin (Pdx). Whether P450cam, once camphor has bound to the active site and the substrate entry channel has closed, opens up upon Pdx binding, during the second electron transfer step, or it remains closed is still a matter of debate. A potential allosteric site for camphor binding has been identified and postulated to play a role in the binding of Pdx. Here, we have revisited paramagnetic NMR spectroscopy data and determined a heterogeneous ensemble of structures that explains the data, provides a complete representation of the P450cam/Pdx complex in solution, and reconciles alternative hypotheses. The allosteric camphor binding site is always present, and the conformational changes induced by camphor binding to this site facilitates Pdx binding. We also determined that the state to which Pdx binds comprises an ensemble of structures that have features of both the open and closed state. These results demonstrate that there is a finely balanced interaction between allosteric camphor binding and the binding of Pdx at high camphor concentrations.

NMR spectroscopy is an indispensably powerful technique for the analysis of biomolecules under ambient conditions, both for structural- and functional studies. However, in practice the complexity of the technique has often frustrated its application by non-specialists. In this paper, we present CcpNmr version-3, the latest software release from the Collaborative Computational Project for NMR, for all aspects of NMR data analysis, including liquid- and solid-state NMR data. This software has been designed to be simple, functional and flexible, and aims to ensure that routine tasks can be performed in a straightforward manner. We have designed the software according to modern software engineering principles and leveraged the capabilities of modern graphics libraries to simplify a variety of data analysis tasks. We describe the process of backbone assignment as an example of the flexibility and simplicity of implementing workflows, as well as the toolkit used to create the necessary graphics for this workflow. The package can be downloaded from www.ccpn.ac.uk/v3-software/downloads and is freely available to all non-profit organisations.

Significance The ubiquitous enzymes called cytochromes P450 catalyze a broad range of chemical reactions using molecular oxygen. For example, in humans, these enzymes are involved in breakdown of foreign compounds, including drugs. The bacterial cytochrome P450cam is thought to open up to allow substrate to enter the active site, and then to close during catalysis to keep reactive intermediates inside. Surprisingly, recent crystal structures suggested that the enzyme is open during the reaction. We have studied the enzyme in solution using paramagnetic NMR spectroscopy, demonstrating that, in fact, the enzyme is closed. This finding indicates that the subtle balance between open and closed is affected by crystallization, which can lead to the wrong conclusions about the protein dynamics.

Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.

The use of paramagnetic NMR data for the refinement of structures of proteins and protein complexes is widespread. However, the power of paramagnetism for protein assignment has not yet been fully exploited. PARAssign is software that uses pseudocontact shift data derived from several paramagnetic centers attached to the protein to obtain amide and methyl assignments. The ability of PARAssign to perform assignment when the positions of the paramagnetic centers are known and unknown is demonstrated. PARAssign has been tested using synthetic data for methyl assignment of a 47 kDa protein, and using both synthetic and experimental data for amide assignment of a 14 kDa protein. The complex fitting space involved in such an assignment procedure necessitates that good starting conditions are found, both regarding placement and strength of paramagnetic centers. These starting conditions are obtained through automated tensor placement and user-defined tensor parameters. The results presented herein demonstrate that PARAssign is able to successfully perform resonance assignment in large systems with a high degree of reliability. This software provides a method for obtaining the assignments of large systems, which may previously have been unassignable, by using 2D NMR spectral data and a known protein structure.

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.