Internal electron transfer and structural dynamics of cd(1) nitrite reductase revealed by laser CO photodissociation

Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reduc tase (Pa-NiR). We have measured the (d(1)-->c) internal eT rate for the wil d-type protein and a, site-directed mutant (Pa-NiR H327A) which has a subst itution in the d(1)-heme binding pocket; we found the rate of eT to be fast , k(eT) = 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 t he 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 wit h a complex time course that: may be described by two exponential processes (k(1) = 3 x 10(4) s(-1) and k(2) = 0.25 x 10(4) s(-1)), These relaxations do not have a kinetic difference spectrum characteristic of CO recombinatio n; and therefore we conclude that Pa-NiR undergoes structural rearrangement s 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 act ive site alters the CO diffusion properties of the protein, Probably reduci ng steric hindrance. CO rebinding to the:wild-type mixed valence enzyme (c( 3+)d(1)(2+)) which is very slow (k = 0.25 s(-1)) is proposed td be rate-lim ited by the c --> d(1) internal eT event, involving the oxidized dl-heme wh ich has a structure characteristic of the fully oxidized and partially oxid ized Pa-NiR.