SummaryProtein design provides an ultimate test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. While progress has been made in designing proteins that mimic native proteins structurally1–3, it is more difficult to design functional proteins4–8. In comparison to recent successes in designing non-metalloproteins4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes5,8,11–20, since protein metal binding sites are much more varied than non-metal containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal binding site properties in silico, as many of the parameters for metal binding sites, such as force fields are ill-defined. Therefore, the successful design of a structural and functional metalloprotein will greatly advance the field of protein design and our understanding of enzymes. Here, we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a heme/non-heme FeB center that is remarkably similar to that in the crystal structure. This designed protein also exhibits NOR activity. This is the first designed protein that models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

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Hydrogen exchange (HX) rates and midpoint potentials (E m ) of variants of cytochromes c from Pseudomonas aeruginosa (Pa cyt c 551 ) and Hydrogenobacter thermophilus (Ht cyt c 552 ) have been characterized toward developing an understanding of the impact of properties of the Cys-X-X-CysHis pentapeptide c-heme attachment motif (CXXCH) on heme redox potential. Despite structural conservation of the CXXCH motif, Ht cyt c 552 exhibits low protection from HX for amide protons within this motif relative to Pa cyt c 551 . Site-directed mutants have been prepared to determine the structural basis for and functional implications of these variations in HX behavior. The double mutant Ht-M13V/K22M displays suppressed HX within the CXXCH motif as well as decreased E m (by 81 mV), whereas the corresponding double mutant of Pa cyt c 551 (V13M/M22K) exhibits enhanced HX within the CXXCH pentapeptide and a modest increase in E m (by 30 mV). The changes in E m correlate with changes in axial His chemical shifts in the ferric proteins reflecting extent of histidinate character. Thus the mobility of the CXXCH pentapeptide is found to impact the His-Fe(III) interaction and therefore heme redox potential.Electron transfer reactions involving iron-protoporphyrin IX (heme) are central to fundamental biological processes such as respiration, redox catalysis, sensing, and signaling (1-5). A key parameter determining energetics and kinetics of electron transfer is the redox potential (1), thus, much emphasis has been placed on understanding the role of protein structure in tuning heme redox potential. Two fundamental features known to have a substantial influence on heme redox potentials are the nature of the ligands coordinated to the metal and the burial of the heme in the hydrophobic protein core. Nature alters the electron donating properties of the coordinating ligands through choice of ligands (6), modulating metal-ligand bond strength (6-12), varying coordination geometry (5), and hydrogen bonding to ligands (13)(14)(15). The encapsulation of the heme within a protein's interior also is significant for determining potential, as the hydrophobic environment favors the ferrous state over the ferric (7,8,10,16,17). Although there have been many studies of the effects of static polypeptide structure on heme-ligand interactions and on heme burial, the role of protein mobility has received less attention. Protein motions may indeed be important as they could influence metal-ligand interactions (15,18,19) and solvent exposure. † This work supported by National Institutes of Health Grant GM63170 (K.L.B.), a Fellowship from the Alfred P. Sloan Foundation (K.L.B.), and National Science Foundation Grant MCB-0546323 (S.J.E.).*To whom correspondence should be addressed: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216. Telephone: (585) Here, we investigate the effects of structural fluctuations of the c-heme motif of cytochrome c (cyt c 1 ) on redox potential. The c-type heme is characterized by its covalen...

The heme group in paramagnetic (S ‫؍‬ 1͞2) ferricytochromes c typically displays a markedly asymmetric distribution of unpaired electron spin density among the heme pyrrole ␤ substituents. This asymmetry is determined by the orientations of the heme axial ligands, histidine and methionine. One exception to this is ferricytochrome c552 from Hydrogenobacter thermophilus, which has similar amounts of unpaired electron spin density at the ␤ substituents on all four heme pyrroles. Here, determination of the orientation of the magnetic axes and analysis of NMR line shapes for H. thermophilus ferricytochrome c 552 is performed. These data reveal that the unusual electronic structure for this protein is a result of fluxionality of the heme axial methionine. It is proposed that the ligand undergoes inversion at the pyramidal sulfur, and the rapid interconversion between two diastereomeric forms results in the unusual heme electronic structure. Thus a fluxional process for a metal-bound amino acid side chain has now been identified.

Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c(552).