Table 3. Substrate reducing activities upon FeMoco addition.

	Activities
	C2H4 formation under C2H2/Ar		H2 formation under Ar		NH3 formation under N2		H2 formation under N2
Protein	Per mg of protein	Per nmol of αβ-dimer	Per mg of protein	Per nmol of αβ-dimer	Per mg of protein	Per nmol of αβ-dimer	Per mg of protein	Per nmol of αβ-dimer
ΔnifB Av1*	1,623 ± 15	173 ± 17 (100)	1,723 ± 82	184 ± 9 (100)	640 ± 23	68 ± 2 (100)	357 ± 43	38 ± 2 (100)
ΔnifH Av1	0	0 (0)	0	0 (0)	0	0 (0)	0	0 (0)
ΔnifH Av1′	148 ± 32	16 ± 3 (9)	172 ± 7	18 ± 1 (10)	85 ± 25	9 ± 3 (13)	26 ± 6	3 ± 1 (8)
ΔnifB Av1v†‡	66 ± 4	11 ± 1 (6)	183 ± 11	29 ± 2 (16)	15 ± 1	2 ± 0.2 (3)	188 ± 23	30 ± 2 (79)

It needs to be noted that IDS-oxidized, FeMoco-reconstituted ΔnifH Av1′ or ΔnifB Av1^v does not show the g = 11.8 parallel-mode EPR signal (Fig. 2C), which is typical for the IDS-oxidized P²⁺ state of the P-cluster. This observation indicates the presence of active P-cluster variants in ΔnifH Av1′ and ΔnifB Av1^v after FeMoco reconstitution. Activities are expressed in nmol per min per mg of protein and nmol per min per nmol of complete αβ-dimer. The percentages of activities relative to those per nmol of αβ-dimer of ΔnifB Av1 are given in parentheses. The lower detection limits were 0.01, 0.02. 0.001, and 0.02 nmol per min per mg of protein for C₂H₄ formation under C₂H₂/Ar, H₂ formation under Ar, NH₃ formation under N₂, and H₂ formation under N₂, respectively. ΔnifB Av1, ΔnifH Av1, ΔnifH Av1′, and ΔnifB Av1^v do not show any substrate reducing activities without the addition of isolated FeMoco.

^*Specific activities of FeMoco-reconstituted ΔnifB Av1 are not appreciably different from those of WT Av1 expressed by AvDJ1141 (data not shown).

^†Isolation of FeVco from WT Av1^v on a scale that is required for our experiments has not been reported so far. Therefore, it is not possible at this point to provide any data of substrate reducing activities of FeVco-reconstituted ΔnifB Av1^v. Nonetheless, a FeMoco-containing ΔnifB Av1^v “hybrid” could be formed, and its specific activities were compared with the activities of fully FeMoco-reconstituted ΔnifB Av1. In this context it should be noted that the FeMoco-containing Av1 shows considerably higher substrate reducing activities than FeVco-containing Av1^v (26, 27). Therefore, the FeMoco-reconstituted ΔnifB Av1^v will show higher percentages of activation if Av1^v is used as a standard and may represent an almost fully activated FeMoco-Av1^v hybrid. However, a conclusive assessment of the percentages of activation of ΔnifB Av1^v is only feasible when a sufficient amount of isolated FeVco is available.

^‡The FeMoco reconstitution assay contained only purified αβ₂-trimeric ΔnifB Av1^v and isolated FeMoco. An αβ₂-trimeric subunit composition of FeMoco-reconstituted ΔnifB Av1^v was confirmed by molecular weight determination of the protein on the Sephacryl S-200 HR gel filtration column (data not shown).