Rational design of a structural and functional nitric oxide reductase.

Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes.

Heme attachment motif mobility tunes cytochrome c redox potential.

Hydrogen exchange (HX) rates and midpoint potentials (Em) of variants of cytochrome c from Pseudomonas aeruginosa (Pa cyt c551) and Hydrogenobacter thermophilus (Ht cyt c552) have been characterized in an effort to develop an understanding of the impact of properties of the Cys-X-X-Cys-His pentapeptide c-heme attachment (CXXCH) motif on heme redox potential. Despite structural conservation of the CXXCH motif, Ht cyt c552 exhibits a low level of protection from HX for amide protons within this motif relative to Pa cyt c551. Site-directed mutants have been prepared to determine the structural basis for and functional implications of these variations on HX behavior.

Effects of heme pocket structure and mobility on cytochrome c stability.

Unfolding thermodynamics of a thermophilic cytochrome c552 from Hydrogenobacter thermophilus (Ht cyt c552) and its mesophilic homologue from Pseudomonas aeruginosa (Pa cyt c551) as well as two heme pocket point mutants (Ht-Q64N and Pa-N64Q) are characterized by determination of protein stability curves (plots of unfolding free energy, DeltaG, vs T). These proteins show revealing differences in heme pocket hydrogen bonding and mobility. It previously has been shown that Asn64 in Pa cyt c551 and in Ht-Q64N interacts with the heme axial Met to fix it in a single conformation [Wen, X.

Catalytic reduction of NO to N2O by a designed heme copper center in myoglobin: implications for the role of metal ions.

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV-vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO.mol CuBMb-1.

Heme axial methionine fluxionality in Hydrogenobacter thermophilus cytochrome c552.

The heme group in paramagnetic (S = 1/2) ferricytochromes c typically displays a markedly asymmetric distribution of unpaired electron spin density among the heme pyrrole beta substituents. This asymmetry is determined by the orientations of the heme axial ligands, histidine and methionine. One exception to this is ferricytochrome c(552) from Hydrogenobacter thermophilus, which has similar amounts of unpaired electron spin density at the beta substituents on all four heme pyrroles.

Backbone dynamics and hydrogen exchange of Pseudomonas aeruginosa ferricytochrome c(551).

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c(551) dynamics based on (15)N R(1), (15)N R(2), and [(1)H]-(15)N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (< S(2)>=0.924+/-0.

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c.

Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation.

Characterization of Hydrogenobacter thermophilus cytochromes c(552 )expressed in the cytoplasm and periplasm of Escherichia coli.

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.