Structural Basis for the Mechanism of Ca2+ Activation of the Di-Heme Cytochrome c Peroxidase from Pseudomonas nautica 617

Abstract

Cytochrome c peroxidase (CCP) catalyses the reduction of H(2)O(2) to H(2)O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca(2+) and was refined at 2.2 A resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca(2+) dependent and was crystallized at pH 5.3 and refined at 2.4 A resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

Structural and Mutagenesis Studies on the Cytochrome c Peroxidase from Rhodobacter capsulatus Provide New Insights into Structure-Function Relationships of Bacterial Di-heme Peroxidases

“…1B). This geometry is analogous to that observed in the PaCCP and PnCCP-OUT structures (8,10) but is lacking two additional water molecules between the calcium and the propionate of the high potential heme group that help complete the distorted pentagonal bipyramidal geometry seen in the other two structures. As in PaCCP and PnCCP, the charge of the calcium ion in RcCCP is not countered by negatively charged groups, suggesting that it can act as a modulator of electron transfer between the two heme domains.…”

“…In the LP-heme, His-74 would be replaced by Met-118, whereas the HP-heme would lose its sixth ligand (Met-278). Structural data from several bacterial cytochrome c peroxidases and, in particular, recent studies on the P. nautica enzyme favor the first model (10). In that study the structure of the active form of PnCCP, termed PnCCP-OUT, reveals that the N-terminal heme group is penta-coordinated.…”

“…The high potential heme, with a midpoint potential of ϩ270 mV, therefore, functions as the electron transferring heme, whereas the low potential heme has a midpoint potential between Ϫ190 and Ϫ310 mV (7) and is the peroxidatic center. Structures of the bacterial cytochrome c peroxidases isolated from Pseudomonas aeruginosa (Pa) (8), N. europaea (9), and Pseudomonas nautica (Pn) (10) have already been determined. The structure of the P. aeruginosa enzyme is that of the completely oxidized, inactive enzyme with bound calcium.…”

“…That from N. europaea is also of the completely oxidized enzyme, but the peroxidase retains activity in this state. Recently, two distinct structural snapshots of CCP from P. nautica became available as the result of the serendipitous reduction of the C-terminal heme group by synchrotron X-rays used for the structural studies (10). The inactive form did not contain Ca 2ϩ and was captured in a closed conformation (IN-form) where the N-terminal peroxidatic heme was coordinated by six ligands, thus blocking the peroxidatic reaction from taking place.…”

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…Actually, in bacterial enzymes containing more than one domain, such as bacterial cytochrome c peroxidase and nitrite reductase cytochrome cd 1 , it has been observed that reduction of the redox center in one of the domains implies a structural change in the protein [62][63][64][65]. Therefore, similar behavior cannot be ruled out for this enzyme and further experiments are currently in progress in our laboratory to test this hypothesis.…”