Christopher A. Bonagura

Conversion of an Engineered Potassium-binding Site into a Calcium-selective Site in Cytochrome c Peroxidase

We have previously shown that the K+ site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A., Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107–6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538–5545). All other peroxidases bind Ca2+ rather than K+. Using the K+-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca2+ selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na+, is bound instead of Ca2+. This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca2+ binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp191 radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca2+ more effectively than K+, demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.

Structure of C42D Azotobacter vinelandii FdI A Cys-X-X-Asp-X-X-Cys MOTIF LIGATES AN AIR-STABLE [4Fe-4S]2+/+ CLUSTER

All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S]2+/+ clusters that can be easily and reversibly converted to [3Fe-4S]+/0 clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S]2+/+ clusters that cannot be easily interconverted with [3Fe-4S]+/0 clusters. In this study we changed the central cysteine of the Cys39-X-X-Cys42-X-X-Cys45of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S]2+/+ cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S]2+/+ cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S]+ cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.

Azotobacter vinelandii Ferredoxin I A SEQUENCE AND STRUCTURE COMPARISON APPROACH TO ALTERATION OF [4Fe-4S]2+/+ REDUCTION POTENTIAL

The reduction potential (E 0′) of the [4Fe-4S]2+/+ cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is ∼200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences inE 0′ do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S]2+/+clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S]2+/+ cluster with a very negativeE 0′ similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E 0′ clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S]2+/+cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higherE 0′ P. asaccharolyticus ferredoxin all led to increases in E 0′ for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH–S bond between the introduced Ser side chain and the Sγ atom of Cys ligand 42 and an accompanying movement of water.

Loop Stability in the Engineered Potassium Binding Site of Cytochrome c Peroxidase

Abstract The Trp 191 containing flexible loop of cytochrome c peroxidase (CcP) exists in equilibrium between open and closed conformers. The open conformer creates a cavity in the loop, which enables it to bind protonated forms of imidazole derivatives such 1,2-dimethylimidazolium (DMI). In the present study we have engineered the K + binding site into CcP and find the equilibrium of the conformer shifted in favor of the open form probably due to electrostatic destabilization. Subsequent changing of a hinge residue in the loop, Asn 195 , to Pro stabilizes the loop in the presence of the bound K + .