4 research outputs found
Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica
Biochemistry, 2011, 50 (20), pp 4251ā4262
DOI: 10.1021/bi101605pRespiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. MoĢssbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the MoĢssbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ā¼ 6 and g ā¼ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ā¼ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction
Combining a Commercial Mixer with a Wall-Tube Electrode Allows the Arbitrary Control of Concentrations in Protein Film Electrochemistry
Protein film electrochemistry is a technique in which
an enzyme
is immobilized on an electrode in a configuration that allows following
the changes in turnover frequency as a response to changes in the
experimental conditions. Insights into the reactivity of the enzyme
can be obtained by quantitatively modeling such responses. As a consequence,
the more the technique allows flexibility in changing conditions,
the more useful it becomes. The most commonly used setup, based on
the rotating disc electrode, allows easy stepwise increases in the
concentration of nongaseous substrates, or exposure to constant concentration
of dissolved gas, but does not permit to easily decrease the concentration
of nongaseous substrates, or to change the concentration of dissolved
gas in a stepwise fashion. To overcome the limitation by mass transport
of the substrate toward the electrode when working with fast enzymes,
we have designed another kind of electrochemical cell based on the
wall-tube electrode (WTE). We demonstrate here that by using a system
combining two syringe pumps, a commercial mixer, and the WTE, it is
possible to change the concentration of species in a stepwise fashion
in all directions, opening new possibilities to study redox enzymes.
As a proof of concept, this device was applied to the study of the
electrochemical response of the cytochrome c nitrite
reductase of Desulfovibrio desulfuricans
Spectroscopic Evidence for and Characterization of a Trinuclear Ferroxidase Center in Bacterial Ferritin from Desulfovibrio vulgaris Hildenborough
Ferritins are ubiquitous and can be found in practically
all organisms that utilize Fe. They are composed of 24 subunits forming
a hollow sphere with an inner cavity of ā¼80 Ć
in diameter.
The main function of ferritin is to oxidize the cytotoxic Fe<sup>2+</sup> ions and store the oxidized Fe in the inner cavity. It has been
established that the initial step of rapid oxidation of Fe<sup>2+</sup> (ferroxidation) by H-type ferritins, found in vertebrates, occurs
at a diiron binding center, termed the ferroxidase center. In bacterial
ferritins, however, X-ray crystallographic evidence and amino acid
sequence analysis revealed a trinuclear Fe binding center comprising
a binuclear Fe binding center (sites A and B), homologous to the ferroxidase
center of H-type ferritin, and an adjacent mononuclear Fe binding
site (site C). In an effort to obtain further evidence supporting
the presence of a trinuclear Fe binding center in bacterial ferritins
and to gain information on the states of the iron bound to the trinuclear
center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric
amounts of Fe<sup>2+</sup>, and the products were characterized by
MoĢssbauer and EPR spectroscopy. Four distinct Fe species were
identified: a paramagnetic diferrous species, a diamagnetic diferrous
species, a mixed valence Fe<sup>2+</sup>Fe<sup>3+</sup> species, and
a mononuclear Fe<sup>2+</sup> species. The latter three species were
detected in the wild-type DvFtn, while the paramagnetic diferrous
species was detected in the E130A variant. These observations can
be rationally explained by the presence of a trinuclear Fe binding
center, and the four Fe species can be properly assigned to the three
Fe binding sites. Further, our spectroscopic data suggest that (1)
the fully occupied trinuclear center supports an all ferrous state,
(2) sites B and C are bridged by a Ī¼-OH group forming a diiron
subcenter within the trinuclear center, and (3) this subcenter can
afford both a mixed valence Fe<sup>2+</sup>Fe<sup>3+</sup> state and
a diferrous state. Mechanistic insights provided by these new findings
are discussed and a minimal mechanistic scheme involving OāO
bond cleavage is proposed