Atomic resolution crystal structures, EXAFS, and quantum chemical studies of rusticyanin and its two mutants provide insight into its unusual properties.

Rusticyanin from the extremophile Thiobacillus ferrooxidans is a blue copper protein with unusually high redox potential and acid stability. We present the crystal structures of native rusticyanin and of its Cu site mutant His143Met at 1.27 and 1.10 A, respectively. The very high resolution of these structures allows a direct comparison with EXAFS data and with quantum chemical models of the oxidized and reduced forms of the proteins, based upon both isolated and embedded clusters and density functional theory (DFT) methods. We further predict the structure of the Cu(II) form of the His143Met mutant which has been experimentally inaccessible due to its very high redox potential. We also present metrical EXAFS data and quantum chemical calculations for the oxidized and reduced states of the Met148Gln mutant, this protein having the lowest redox potential of all currently characterized mutants of rusticyanin. These data offer new insights into the structural factors which affect the redox potential in this important class of proteins. Calculations successfully predict the structure and the order of redox potentials for the three proteins. The calculated redox potential of H143M ( approximately 400 mV greater than native rusticyanin) is consistent with the failure of readily available chemical oxidants to restore a Cu(II) species of this mutant. The structural and energetic effects of mutating the equatorial cysteine to serine, yet to be studied experimentally, are predicted to be considerable by our calculations.

Insights into redox partner interactions and substrate binding in nitrite reductase from Alcaligenes xylosoxidans: crystal structures of the Trp138His and His313Gln mutants.

Dissimilatory nitrite reductase catalyses the reduction of nitrite to nitric oxide within the key biological process of denitrification. We present biochemical and structural results on two key mutants, one postulated to be important for the interaction with the partner protein and the other for substrate entry. Trp138, adjacent to one of the type-1 Cu ligands, is one of the residues surrounding a small depression speculated to be important in complex formation with the physiological redox partners, azurin I and II. Our data reveal that the Trp138His mutant is fully active using methyl viologen as an artificial electron donor, but there is a large decrease in activity using azurin I. These observations together with its crystal structure at a high resolution of 1.6 A confirm the importance of Trp138 in electron transfer and thus in productive interaction with azurin. A "hydrophobic pocket" on the protein surface has been identified as the channel through which nitrite may be guided to the catalytic type-2 Cu site. Glu133 and His313 at the opening of the pocket are conserved among most blue and green copper nitrite reductases (CuNiRs). The failure to soak the substrate into our high-resolution crystal form of native and mutant CuNiRs has been linked to the observation of an extraneous poly(ethylene glycol) (PEG) molecule interacting with His313. We present the crystal structure of His313Gln and the substrate-bound mutant at high resolutions of 1.65 and 1.72 A, respectively. The observation of the substrate-bound structure for the His313Gln mutant and inhibitory studies with PEG establishes the role of the hydrophobic pocket as the port of substrate entry.