Spectroscopic, Kinetic, and Electrochemical Characterization of Heterologously Expressed Wild-Type and Mutant Forms of Copper-Containing Nitrite Reductase from Rhodobacter sphaeroides 2.4.3

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• 作者：Kenneth Olesen ; Andrei Veselov ; Yiwei Zhao ; Yousheng Wang ; Birgit Danner ; Charles P. Scholes ; and James P. Shapleigh
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：April 28, 1998
• 年：1998
• 卷：37
• 期：17
• 页码：6086 - 6094
• 全文大小：154K
• 年卷期：v.37,no.17(April 28, 1998)
• ISSN：1520-4995

文摘

We report the development of a high-yield heterologous expressionsystem for the copper-containing nitrite reductase from a denitrifying variant ofRhodobacter sphaeroides. Typical yieldsofwild-type protein are 20 mg L^-1, which can befully loaded with copper. Nitrite reductase containsanunusual blue-green Type 1 copper center with a redox/electron transferfunction and a nearby Type 2center where nitrite binds and is reduced to nitric oxide. Thewild-type enzyme was characterized by:(1) its blue-green Type 1 optical spectrum; (2) its EPR spectrumshowing rhombic character to its Type1 center and nitrite perturbation to its Type 2 center; (3) its 247-mVType 1 midpoint potential which islow relative to other Type 1 centers; and (4) its kinetics as measuredby both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored byEPR in the absence of nitrite wasbelow 200 mV so that reduction of the Type 2 center by the Type 1center in the absence of nitrite is notenergetically favored. The mutation M182T in which the methionineligand of Type 1 copper was changedto a threonine resulted in a blue rather than blue-green Type 1 center,a midpoint potential that increasedby more than 100 mV above that of the wild-type Type 1 center, and asomewhat reduced nitrite reductaseactivity. The blue color and midpoint potential of M182T arereminiscent of plastocyanin, but the Type1 cupric HOMO ground-state electronic g value and copperhyperfine properties of M182T (as well ascysteine and histidine ENDOR hyperfine properties; see next paper) wereunchanged from those of theblue-green native Type 1 center. His²⁸⁷ is a residuein the Type 2 region whose imidazole ring wasthought to hydrogen bond to the Type 2 axial ligand but not directly toType 2 copper. The mutationH287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPRspectrum (as well as ENDORspectra; see next paper) which were no longer sensitive to the presenceof nitrite.