Atomic Resolution Crystal Structures, EXAFS, and Quantum Chemical Studies of Rusticyanin and Its Two Mutants Provide Insight into Its Unusual Properties^,

Abstract

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.

The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism

“…With respect to Fe oxidation, a putative rusticyanin (Msed1206), the locus 3 respiratory cluster (Msed0500 to Msed0504) expected to be up-regulated on Fe(II), and a unique putative quinol oxidase fusion protein (cytochrome b N-terminal and Rieske protein C-terminal domains) were highly transcribed in the presence of FeSO 4 . Several fox genes of locus 4 (specifically Msed0477 to Msed0480) were also highly transcribed on FeSO 4 , although levels in the presence of YE alone were at the upper end of the dynamic response range of the array. The exception to this was subunit I of the terminal oxidase (SoxB; Msed0484), which was more highly transcribed in the presence of FeSO 4 ϩYE.…”

Identification of Components of Electron Transport Chains in the Extremely Thermoacidophilic Crenarchaeon Metallosphaera sedula through Iron and Sulfur Compound Oxidation Transcriptomes

“…A blue copper protein, rusticyanin, is implicated in electron transport from iron oxidation, and one version of this protein has been extensively characterized in A. ferrooxidans (4,51,53). The M. sedula genome encodes four blue copper protein-like sequences: two sulfocyanins (Msed0323 and Msed0826) and two rusticyanins (2).…”

NMR Hyperfine Shifts in Blue Copper Proteins: A Quantum Chemical Investigation

“…The Protein Data Bank (PDB) structure file numbers and their corresponding crystallographic resolutions were as follows: 1AAC (1.31 Å) for Am; 23 1NWP (1.60 Å) for Az; 24 1PLC (1.33 Å) for Pc; 25 1BQK (1.35 Å) for Pa; 26 1JER (1.60 Å) for St; 27 and 2CAK (1.27 Å) for Rc. 28 These are generally the highest resolution structures for these proteins, except for Am and Az, where two new higher resolution structures (i.e., 2OV0, 0.75 Å; 29 2CCW, 1.13 Å; 30 respectively) were recently deposited in the PDB and were also investigated for comparison. We also investigated 4AZU (1.90 Å) 31 for Az, to address the possible effects of species differences on the NMR hyperfine shift predictions.…”