Search

Soluble guanylate cyclase (sGC), the mammalian receptor for nitric oxide (NO), is a heme protein with a histidine as the proximal ligand. Formation of a five-coordinate heme-NO complex with the associated Fe-His bond cleavage is believed to trigger a conformational change that activates the enzyme and transduces the NO signal. Cytochrome c' (cyt c') is a protobacteria heme protein that has several similarities with sGC, including the ability to form a five-coordinate NO adduct and the fact that it does not bind oxygen. Recent crystallographic characterization of cyt c' from Alcaligenes xylosoxidans (AXCP) has yielded the discovery that exogenous ligands are able to bind to the Fe center from either side of the porphyrin plane. In this paper, we explore the molecular basis of the NO interaction with AXCP using hybrid quantum-classical simulation techniques. Our results suggest that Fe-His bond breaking depends not only on the iron-histidine bond strength but also on the existence of a local minimum conformation of the protein with the histidine away from the iron. We also show that AXCP is a useful paradigm for NO interaction with heme proteins, particularly regarding the activation/deactivation mechanism of sGC. The results presented here fully support a recently proposed model of sGC activation in which NO is not only the iron ligand but also catalyzes the activation step.

Loop-contraction mutagenesis has been applied to the cupredoxin pseudoazurin to introduce the active-site loop of amicyanin. The mutation has a limited effect on the spectroscopic properties, and therefore structure, of the cupric protein. The loop contraction results in the increase of the pKa for the detachable His ligand of pseudoazurin by two pH units, similar to the value as found in amicyanin.

The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T. Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom). These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site. The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector). It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond. The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand. It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.