title: Internal electron transfer and structural dynamics of cd1 nitrite reductase revealed by laser CO photodissociation.
abstract: Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR). We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively. We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1). These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO. The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance. CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.
authors.affiliation: Dipartimento di Scienze Biochimiche, Centro di Biologia Moleculare del CNR, Università di Roma La Sapienza, Italy.
authors.forename: E K
authors.initials: EK
authors.lastname: Wilson
authors.forename: A
authors.initials: A
authors.lastname: Bellelli
authors.forename: S
authors.initials: S
authors.lastname: Liberti
authors.forename: M
authors.initials: M
authors.lastname: Arese
authors.forename: S
authors.initials: S
authors.lastname: Grasso
authors.forename: F
authors.initials: F
authors.lastname: Cutruzzolà
authors.forename: M
authors.initials: M
authors.lastname: Brunori
authors.forename: P
authors.initials: P
authors.lastname: Brzezinski
mesh_terms: D000409:Alanine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism; D003574:Cytochrome c Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D007834:Lasers; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010782:Photolysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013053:Spectrophotometry; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D003574:Cytochrome c Group; D003580:Cytochromes; D006639:Histidine; D002248:Carbon Monoxide; C019315:cytochrome cd1; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing; D000409:Alanine