Search for novel redox groups in mitochondrial NADH:ubiquinone oxidoreductase (complex I) by diode array UV/VIS spectroscopy

Abstract

The proton-translocating NADH:ubiquinone oxidoreductase of mitochondria (complex I) is a large L-shaped multisubunit complex. The peripheral matrix arm contains one FMN and a number of iron-sulfur (FeS) clusters and is involved in NADH oxidation and electron transfer to the membrane intrinsic arm. There, following a yet unknown mechanism, the redox-driven proton translocation and the ubiquinone reduction take place. Redox groups that would be able to link electron transfer with proton translocation have not been found so far in the membrane arm. We searched for such groups in complex I isolated from Neurospora crassa. Under anaerobic conditions, the preparation was analyzed in different redox states by means of UV/VIS and EPR spectroscopy. Absorption bands in the UV/VIS redox difference spectra were found which cannot be attributed to the FMN or the EPR detectable FeS clusters. The existence of two novel groups is postulated and their possible locations in the electron pathway and their roles in proton translocation are discussed.

Disruption of iron-sulphur cluster N2 from NADH:ubiquinone oxidoreductase by site-directed mutagenesis

“…The redox difference spectra obtained by subtracting the spectrum of complex I before and after this accelerated reoxidation is dominated by negative absorbances at 325 and 425 nm ( Figure 5, trace B), attributed to clusters N6a and N6b [7]. Further reoxidation of complex I involving cluster N2 and redox group X is very slow [12]. The UV\visible spectrum of partly reoxidized wild-type complex I in this state, after subtraction of a spectrum of the untreated oxidized complex I, is characterized by a positive absorption band around 300 nm, and a broad negative absorption band around 430 nm ( Figure 5, trace A).…”

“…As the UV\visible spectra are characterized by broad overlapping absorptions not allowing a differentiation of redox groups, UV\visible redox difference spectra were taken. The redox difference spectra of complex I display absorption bands attributable to FMN and iron-sulphur clusters as well as additional absorption bands that were considered to be caused by redox group X [7,12]. To compare redox difference spectra of wild-type and mutant complex I, reduction of complexes by different amounts of NADH and reoxidation by oxygen were monitored by UV\visible spectroscopy.…”

“…It has been suggested therefore that cluster N2 is the electron donor to quinone and is directly involved in proton translocation [10,11]. Recently, the existence of an additional redox group in complex I with a midpoint potential above k100 mV was proposed [12,13]. The molecular nature of this redox group is unknown and has been designated redox group X for the time being.…”

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

“…For some purified eukaryotic, non-mammalian enzymes the Fe:FMN ratio's are 27.5 (Torulopsis utilis (Tottmar and Ragan 1971)), 28-38 (N. crassa (Schulte et al 1998)), 33.2 (Pichia pastoris (Bridges et al 2009)) and 30.4 (Pichia angusta (Bridges et al 2009)). The values for the purified Y. lipolytica enzyme have not been reported (Djafarzadeh et al 2000;Kashani-Poor et al 2001a).…”

Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins

“…However, the mechanism of proton transfer is not understood. Various proposals have been made, including a conformational coupling mechanism (33)(34)(35), a mechanism related to the Qcycle that operates in complex III (36), and a mechanism depending on the possible presence of an unidentified cofactor in the membrane domain of the enzyme (37,38). If this cofactor exists, it is not attached to any of the six ND subunits that have been analyzed (assuming that it is stable at pH 3.7), nor to any of the 13 nuclear-encoded subunits that contribute to the membrane domain.…”