The reactions of nitric oxide and nitrite with hemerythrin

The reactions of NO and NO(,2)('-) with the nonheme oxygen carrier, hemerythrin (Hr), were studied in order to shed further light on two physiological functions of Hr: reversible oxygenation and auto-oxidation;A green-colored nitric oxide adduct of deoxyHr (deoxyNO) results from the reactions of NO with deoxyHr and oxyHr. The quadrupole doublets observed in the Mossbauer spectrum are assigned to an S = 2 Fe('2+) center and an S = 3/2 FeNO ('7) site. Low-temperature broadening of both doublets and the occurrence of a novel EPR signal indicate that these two sites are antiferromagnetically coupled to produce an S(,eff) = 1/2 ground state. Identification of (nu)(Fe-NO) and (delta)(Fe-N-O) in the resonance Raman spectrum is consistent with end-on coordination of NO to iron. The downward shift of (delta)(Fe-N-O) in D(,2)O indicates that the ligand engages in hydrogen-bonding with the (mu)-hydroxo group and is consistent with a bent Fe-N-O geometry. Formulation of the oxidation states as Fe('2+)Fe('3+)NO('-) is analogous to that of a putative Fe('2+)Fe('3+)O(,2)('-) intermediate of the oxygenation reaction. As such, these results suggest that during oxygenation, the iron atoms are antiferromagnetically coupled prior to formal oxidation of the second iron atom;When NO is added to deoxyF('-), a more stable adduct (deoxyF('-)NO) results. Evidence suggests that a (mu)-fluoro bridge has replaced the (mu)-hydroxo bridge of deoxyNO. The relative affinities of deoxyHr, deoxyF('-), and semi-metHr for NO could provide a basis for delineation of the factors that affect the affinity of Hr for oxygen;The reaction of deoxyHr with NaNO(,2) occurs in two phases. The first phase (k(,1) = 2.10 M('-1)s('-1) at pH 6.58) produces a brown, EPR-silent product. This product, identified as semi-metNO, was also prepared by adding NO to semi-metHr. The second phase (k(,2) = 6.11 x 10('-4) s('-1)) at pH 6.58) is attributed to rate-limiting loss of NO followed by the rapid binding of NO(,2)('-). The final product is a paramagnetic nitrite adduct of semi-metHr, semi-metNO(,2)('-). In contrast to all the other oxidants used to date (besides O(,2)), nitrite reacts directly at the iron center of deoxyHr in an "inner sphere" fashion. Semi-metNO(,2)('-) is also detected when oxyHr is auto-oxidized to metHr in the presence of nitrite, which suggests that deoxyHr may be an intermediate in auto-oxidation.</p

Evolving the [Myoglobin, Cytochrome <i>b</i>₅] Complex from Dynamic toward Simple Docking: Charging the Electron Transfer Reactive Patch

We describe photoinitiated electron transfer (ET) from a suite of Zn-substituted myoglobin (Mb) variants to cytochrome <i>b</i>₅ (<i>b</i>₅). An electrostatic interface redesign strategy has led to the introduction of positive charges into the vicinity of the heme edge through D/E → K charge-reversal mutation combinations at “hot spot” residues (D44, D60, and E85), augmented by the elimination of negative charges from Mb or <i>b</i>₅ by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners’ total charges (−5 < −<i>q</i>_Mb<i>q</i>_<i>b</i>₅ < 40). The binding affinity (<i>K</i>_a) increases 1000-fold as −<i>q</i>_Mb<i>q</i>_<i>b</i>₅ increases through this range and exhibits a surprisingly simple, exponential dependence on −<i>q</i>_Mb<i>q</i>_<i>b</i>₅. This is explained in terms of electrostatic interactions between a “charged reactive patch” (crp) on each partner’s surface, defined as a compact region around the heme edge that (i) contains the total protein charge of each variant and (ii) encompasses a major fraction of the “reactive region” (Rr) comprising surface atoms with large matrix elements for electron tunneling to the heme. As −<i>q</i>_Mb<i>q</i>_<i>b</i>₅ increases, the complex undergoes a transition from fast to slow-exchange dynamics on the triplet ET time scale, with a correlated progression in the rate constants for intracomplex (<i>k</i>_et) and bimolecular (<i>k</i>₂) ET. This progression is analyzed by integrating the crp and Rr descriptions of ET into the textbook steady-state treatment of reversible binding between partners that undergo intracomplex ET and found to encompass the full range of behaviors predicted by the model. The generality of this approach is demonstrated by its application to the extensive body of data for the ET complex between the photosynthetic reaction center and cytochrome <i>c</i>₂. Deviations from this model also are discussed