2XX1

STRUCTURE OF THE N90S MUTANT OF NITRITE REDUCTASE FROM ALCALIGENES XYLOSOXIDANS complexed with nitrite

Summary for 2XX1

• Entry DOI: 10.2210/pdb2xx1/pdb
• Related: 1BQ5 1GS6 1GS7 1GS8 1HAU 1HAW 1NDT 1OE1 1OE2 1OE3 1WA0 1WA1 1WA2 1WAE 2BO0 2BP0 2BP8 2JFC 2JL0 2JL3 2VM3 2VM4 2VMJ 2VN3 2VW4 2VW6 2VW7 2XWZ 2XX0 2XXF 2XXG
• Descriptor: DISSIMILATORY COPPER-CONTAINING NITRITE REDUCTASE, COPPER (II) ION, NITRITE ION, ... (5 entities in total)
• Functional Keywords: oxidoreductase, proton channel, denitrification
• Biological source: ACHROMOBACTER XYLOSOXIDANS
• Total number of polymer chains: 6
• Total formula weight: 222322.83

Authors

Antonyuk, S.V.,Leferink, N.G.H.,Han, C.,Heyes, D.J.,Rigby, S.E.J.,Hough, M.A.,Eady, R.R.,Scrutton, N.S.,Hasnain, S.S. (deposition date: 2010-11-07, release date: 2011-05-18, Last modification date: 2023-12-20)

Primary citation

Leferink, N.G.H.,Han, C.,Antonyuk, S.V.,Heyes, D.J.,Rigby, S.E.J.,Hough, M.A.,Eady, R.R.,Scrutton, N.S.,Hasnain, S.S.
Proton-Coupled Electron Transfer in the Catalytic Cycle of Alcaligenes Xylosoxidans Copper-Dependent Nitrite Reductase.
Biochemistry, 50:4121-, 2011

PubMed Abstract: We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S--H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ~70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S--H254F variants, where the T1Cu site redox potential is elevated by ∼60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c(551) with Cu NiR has been suggested previously based on a crystal structure of the binary complex.

PubMed: 21469743
DOI: 10.1021/BI200246F

Experimental method

X-RAY DIFFRACTION (3 Å)