The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. We introduce this method here with the first design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assemble a four helix bundle, position histidines to bis-his ligate hemes, and exploit helical rotation and glutamate burial on heme binding to introduce distal histidine strain and facilitate O2 binding. For stable oxygen binding without heme oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O2 affinities and exchange timescales match natural globins with distal histidines with the remarkable exception that O2 binds tighter than CO.
Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A 2 (PLA 2 ) activities, and it alone among mammalian peroxiredoxins can hydrolyze phospholipids. After identifying a potential catalytic triad (S32, H26, D140) from the crystal structure, site-specific mutations were used to evaluate the role of these residues in protein structure and function. The S32A mutation increased Prdx6 a-helical content, whereas secondary structure was unchanged by mutation to H26A and D140A. Lipid binding by wild-type Prdx6 to negatively charged unilamellar liposomes showed an apparent rate constant of 11.2 3 10 6 M 21 s 21 and a dissociation constant of 0.36 mM. Both binding and PLA 2 activity were abolished in S32A and H26A; in D140A, activity was abolished but binding was unaffected. Overoxidation of the peroxidatic C47 had no effect on lipid binding or PLA 2 activity. Fluorescence resonance energy transfer from endogenous tryptophanyls to lipid probes showed binding of the phospholipid polar head in close proximity to S32. Thus, H26 is a site for interfacial binding to the liposomal surface, S32 has a key role in maintaining Prdx6 structure and for phospholipid substrate binding, and D140 is involved in catalysis. This putative catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate to optimize the protein-substrate complex for hydrolysis.-Manevich, Y., K. S. Reddy, T. Shuvaeva, S. I. Feinstein, and A. B. Fisher. Structure and phospholipase function of peroxiredoxin 6: identification of the catalytic triad and its role in phospholipid substrate binding. J. Lipid Res.
A novel electrochemical system has been designed and assembled to study the kinetic activity of cytochrome c oxidase. Gold electrodes coated with 3-mercapto-1-propanol formed the surface for the physisorption of monolayers of cytochrome c and cytochrome c oxidase or a preformed cytochrome c−cytochrome c oxidase complex. The films were investigated by cyclic voltammetry at scanning at rates slow enough to permit near redox equilibrium between electrode and redox protein and hence obtain redox midpoint potentials. Cytochrome c monolayers alone displayed a reversible midpoint potential at pH 8 (E m8 vs NHE) at +240 mV, close to the native cytochrome c value observed in solution. In contrast, oxidase monolayers alone failed to support any detectable redox contact between electrode and protein, implying that the distances between the oxidase redox cofactors in the adsorbed oxidase are too far away from the electrode to promote significant electron transfer rates. However, adsorption of a preformed cytochrome c−cytochrome c oxidase complex promoted effective redox contact, demonstrating electron transfer with an apparent onset halfpoint potential at +225 mV. This effect is consistent with the mandatory requirement for cytochrome c to mediate electrons from the electrode to cytochrome c oxidase and presumably in a way reflecting the physiological pathway. Cyclic voltammetric measurements arranged to determine the rates of electron transfer between electrode and the complex showed that at scan rates up to 50 mV/s, extraordinary kinetic turnover is displayed attributable to the catalysis of oxygen reduction. Thus it is established that the protein complex can be assembled and enable the natural mediation of electron transfer from the electrode by cytochrome c to the enzyme at a rate fast enough for catalysis to be observed and controlled.
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