Reactions of Heme Proteins to Solutions and Crystals

To assess the effects of heme solvation and iron ligation on reduction potentials in c-type cytochromes, we have examined the redox and ligand-binding properties of microperoxidase-8 (MP8). Methionine-, histidine- and amine-coordination to MP8 were found to account for 130, -40 and -10-mV shifts in the Fe(III/II)-potential, respectively. Our finding that reduction potentials increase with decreasing heme-surface exposure suggests that the protein matrix can further tune the reduction potential by 500 mV through water exclusion from the heme pocket. The 410-mV upshift in the cytochrome c (cyt c) potential as the heme cofactor is moved from a highly-solvated environment to the protein interior signals a 10-kcal/mol greater stability of the reduced form. Consequently, there exists a range of denaturant concentrations where Fe(II)-cyt c is folded and Fe(III)-cyt c is unfolded. Electron injection into the oxidized protein in this range triggers the folding reaction. Using NADH as a redox photosensitizer, cyt c folding can be initiated within 100 µs. Our results suggest that the folding of cyt c is rate-limited by ligand-substitution events on the iron center. Due to an increased barrier to ligand substitution, folding of Co(III)-substituted cyt cis 5 orders of magnitude slower than Fe-cyt c. The slow folding kinetics of Co(III)-cyt c have allowed the convenient study of protein dynamics with a variety of spectroscopic techniques, revealing previously unresolved folding pathways involving Lys- and His-misligated populations of the unfolded molecule and extremely long-lived folding intermediates. Factors that control electron flow between proteins are not well understood, owing to uncertainties in the relative orientations and structures of the reactants during the short time that tunneling occurs. To circumvent this ambiguity, we have measured the kinetics of electron transfer (ET) between native and Zn-substituted tuna cyt c molecules in crystals of known structure. ET rates (320 s-1 for *Zn-cyt c → Fe(III)-cyt c; 2000 s-1 for Fe(II)-cyt c → Zn-cyt c+) over a Zn-Fe distance of 24.1 Å closely match those for intraprotein ET over similar donor-acceptor separations. Our results indicate that van der Waals interactions and water mediated H-bonds provide effective electronic coupling across a protein-protein interface.</p

Probing Protein Folding with Substitution-Inert Metal Ions. Folding Kinetics of Cobalt(III)-Cytochrome c

Ligand-substitution processes at the heme strongly influence peptide backbone dynamics during the folding of cytochrome c (cyt c). When cyt c is unfolded with guanidine hydrochloride (GuHCl) at pH 7, one of the axial ligands (Met 80) is replaced by a nitrogenous base from an amino acid residue; this misligation introduces an energy barrier with an associated folding time of several hundred milliseconds. A great deal of evidence points to His 26 or His 33 as the ligand in unfolded horse heart cyt c. Nevertheless, recent studies indicate that other bases (Lys or N-terminus in yeast cyt c) can act as ligands as well. We have found that the substitution-inert heme in the Co(III) derivative of cyt c (Co-cyt c) allows a closer look at the folding kinetics and the ligands in the unfolded form of this protein

Structural Evidence for Asymmetrical Nucleotide Interactions in Nitrogenase

The roles of ATP hydrolysis in electron-transfer (ET) reactions of the nitrogenase catalytic cycle remain obscure. Here, we present a new structure of a nitrogenase complex crystallized with MgADP and MgAMPPCP, an ATP analogue. In this structure the two nucleotides are bound asymmetrically by the Fe-protein subunits connected to the two different MoFe-protein subunits. This binding mode suggests that ATP hydrolysis and phosphate release may proceed by a stepwise mechanism. Through the associated Fe-protein conformational changes, a stepwise mechanism is anticipated to prolong the lifetime of the Fe-protein-MoFe-protein complex and, in turn, could orchestrate the sequence of intracomplex ET required for substrate reduction

Repurposing proteins for new bioinorganic functions

Inspired by the remarkable sophistication and complexity of natural metalloproteins, the field of protein design and engineering has traditionally sought to understand and recapitulate the design principles that underlie the interplay between metals and protein scaffolds. Yet, some recent efforts in the field demonstrate that it is possible to create new metalloproteins with structural, functional and physico-chemical properties that transcend evolutionary boundaries. This essay aims to highlight some of these efforts and draw attention to the ever-expanding scope of bioinorganic chemistry and its new connections to synthetic biology, biotechnology, supramolecular chemistry and materials engineering

Nitrogenase Complexes: Multiple Docking Sites for a Nucleotide Switch Protein

Adenosine triphosphate (ATP) hydrolysis in the nitrogenase complex controls the cycle of association and dissociation between the electron donor adenosine triphosphatase (ATPase) (Fe-protein) and its target catalytic protein (MoFe-protein), driving the reduction of dinitrogen into ammonia. Crystal structures in different nucleotide states have been determined that identify conformational changes in the nitrogenase complex during ATP turnover. These structures reveal distinct and mutually exclusive interaction sites on the MoFe-protein surface that are selectively populated, depending on the Fe-protein nucleotide state. A consequence of these different docking geometries is that the distance between redox cofactors, a critical determinant of the intermolecular electron transfer rate, is coupled to the nucleotide state. More generally, stabilization of distinct docking geometries by different nucleotide states, as seen for nitrogenase, could enable nucleotide hydrolysis to drive the relative motion of protein partners in molecular motors and other systems

Nitrogenase MoFe-Protein at 1.16 Å Resolution: A Central Ligand in the FeMo-Cofactor

A high-resolution crystallographic analysis of the nitrogenase MoFe-protein reveals a previously unrecognized ligand coordinated to six iron atoms in the center of the catalytically essential FeMo-cofactor. The electron density for this ligand is masked in structures with resolutions lower than 1.55 angstroms, owing to Fourier series termination ripples from the surrounding iron and sulfur atoms in the cofactor. The central atom completes an approximate tetrahedral coordination for the six iron atoms, instead of the trigonal coordination proposed on the basis of lower resolution structures. The crystallographic refinement at 1.16 angstrom resolution is consistent with this newly detected component being a light element, most plausibly nitrogen. The presence of a nitrogen atom in the cofactor would have important implications for the mechanism of dinitrogen reduction by nitrogenase

Charting a course for chemistry

To mark the occasion of Nature Chemistry turning 10 years old, we asked scientists working in different areas of chemistry to tell us what they thought the most exciting, interesting or challenging aspects related to the development of their main field of research will be — here is what they said