Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).
The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.
Monari
Stefano
S
Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, I-41100 Modena, Italy.
Battistuzzi
Gianantonio
G
Borsari
Marco
M
Di Rocco
Giulia
G
Martini
Laura
L
Ranieri
Antonio
A
Sola
Marco
M
D002384:Catalysis; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D004566:Electrodes; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D031161:Pseudoalteromonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013816:Thermodynamics
C505222:11-mercaptoundecanoic acid; D003574:Cytochrome c Group; D004800:Enzymes, Immobilized; D005227:Fatty Acids; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C034186:cytochrome C4; D006418:Heme; D006046:Gold